Method of plant genome design, method of creating new cultivar and new cultivar

ABSTRACT

Plant genome design method defines DNA markers M1 to M5, for each target region, DNA marker M2 is at an upstream side of a target region, or upstream thereof, DNA marker M1 is upstream of DNA marker M2, DNA marker M4 is at a downstream side of the target region, or downstream thereof, DNA marker M5 is downstream of DNA marker M4, and DNA marker M3 is in the target region; and designs a genome so that a substitution region, containing the target region, in a chromosome of the original cultivar to be substituted with a chromosome fragment derived from the foreign cultivar has an end on an upstream side between DNA marker M1 and DNA marker M2, and an end on a downstream side of the substitution region between DNA marker M4 and DNA marker M5.

TECHNICAL FIELD

The present invention relates to a method of designing a plant genome bya non-gene recombinant method, which is suitable for improving acultivar, a method of creating a new cultivar using this genome designmethod, a plant individual and a new cultivar created by this method,and a method of discriminating a plant cultivar.

The present application claims priority on Japanese Patent ApplicationNo. 2008-176934 filed on Jul. 7, 2008, the disclosure of which isincorporated by reference herein.

BACKGROUND ART

A population which belongs to the same organism species, but has adifferent character from that of other population in a certain characterdue to different genetic construction is called variety (cultivar). Thatis, even the same kind of plant is different in difficulty of culturing,pest resistance, yield, quality or the like, depending on a cultivar.For this reason, in agricultural crops, particularly, main crops such asrice and wheat, cultivar improvement for obtaining a more excellentcultivar has been conducted from old times. In recent years, thiscultivar improvement has been positively conducted in not only seed andplant suppliers but also public organizations such as country andprefecture. In addition, in order to response to variety of taste ofconsumers in recent years, in horticultural crops such as grasses andflowers in addition to edible crops, a new cultivar having a variety ofcolors and morphologies is vigorously being developed.

Further, in recent years, an attention is paid to a plant source as araw material such as biomass ethanol and the like, and development of anew cultivar having high source efficiency is expected.

With advance of nucleic acid analysis technique or the like in recentyears, genes of a variety of plants such as Arabidopsis, rice, wheat andthe like have been analyzed, and genetic information obtained by thisanalysis has been disclosed. Utilizing the disclosed geneticinformation, cultivar improvement of introducing a foreign species geneinto an original cultivar by a gene recombinant method is performedfrequency. For example, a Hd1 gene encoding a protein derived from aplant having the function of increasing photosensitivity of a plant, anda method of creating a transformed plant with this Hd1 gene introducedtherein, and the like are disclosed (see, for example, Patent document1). However, although cultivar improvement by a gene recombinant methodhas an advantage that a character possessed by a distant species whichcannot be usually mated can be introduced, there is a problem that studyof safety thereof is not necessarily sufficient.

On the other hand, as a method of improving a plant cultivar by anon-gene recombinant method, there are a breeding method by mating and amutation method. As a breeding method by normal mating, there is apedigree breeding method, a population breeding method, a backcrossbreeding method and the like. Alternatively, by combining the backcrossbreeding method and MAS (Marker Assisted Selection) method, cultivarimprovement of introducing a target gene into an original cultivar isalso widely performed. Herein, the MAS method is a method of selectingan individual having an objective character from a population of a crossprogeny obtained by natural mating which has been conducted from oldtimes or artificial mating using a DNA marker linked to a gene encodingan objective character. By performing individual selection using thisDNA marker, an individual having an objective character can be selectedat an early stage such as a seedling stage and the like, and it can savelabor and improve efficiency. As a method of selecting an individualhaving a particular character using such the DNA marker, for example, amethod of determining a genotype of a plant using a DNA marker presentin a region surrounding a sd-1 gene which is a rice semidwarf gene, anda method of testing semidwarf character of a plant using this method aredisclosed (see, for example, Patent document 2).

RELATED DOCUMENTS Patent Document

-   [Patent document 1] Japanese Patent No. 3,660,967-   [Patent document 2] International Publication No. WO 2003/070934

DISCLOSURE OF INVENTION Problem to be Solved by the Invention

However, in the cultivar improvement method using the MAS method, thereis a problem that a progeny individual selected using the DNA marker isnot necessarily an individual having an objective character, and a roleof the DNA marker becomes only assistant selection. This is presumedthat the DNA marker is usually not on a character gene to be introduced,and a distance whose length and direction are unknown is present betweenthis character gene and the DNA marker. For this reason, in the MASmethod, a size of a population of a progeny individual to becharacter-assessed can be reduced, but it is still necessary to furtherassess a character of a progeny individual selected using the DNAmarker.

In addition, in the cultivar improvement method using the MAS method,there is also a problem that a character of a target is improved, butother character is deteriorated. This is presumed, as described above,that since a distance whose length and direction are unknown is presentbetween an introduced character gene and the DNA marker, a region of achromosome fragment to be introduced cannot be controlled, and manyother genes other than an objective character gene are introduced intothe plant.

One of objects of the present invention is to provide a method of plantgenome design and a method of creating new cultivar for controlling asubstitution region with a chromosome fragment derived from a foreigncultivar to be introduced, and creating a new cultivar having a targetcharacter without changing a preferable character possessed by anoriginal cultivar, in the case where improvement in a cultivar isperformed by a non-gene recombinant method.

Means for Solving the Problems

The present inventors have intensively studied in order to solve theabove problems and found that, when a new cultivar in which a targetregion in a chromosome of an original cultivar is substituted with ahomo-chromosome fragment derived from a foreign cultivar is createdusing a chromosome fragment-substituted line, by satisfying thefollowing conditions (I) and (II), a chromosome region of an originalcultivar substituted with this home-chromosome fragment can becontrolled, resulting in completion of the present invention.

(I) A DNA marker M2 is defined at an end on an upstream side of a targetregion, or upstream thereof, a DNA marker M1 is defined upstream of theDNA marker M2, a DNA marker M4 is defined at an end on a downstream sideof the target region, or downstream thereof, a DNA marker M5 is defineddownstream of the DNA marker M4, and a DNA marker M3 is defined in thetarget region.(II) A progeny individual in which an end on an upstream side of achromosome region of an original cultivar to be substituted with achromosome fragment derived from a foreign cultivar to be introduced isbetween DNA markers M1 and M2, and an end on a downstream side of thisregion is between DNA markers M4 and M5, is selected.(1) A plant genome design method of the present invention is a method ofdesigning a plant genome in which a target region in a chromosome of anoriginal cultivar is substituted with a chromosome fragment derived fromthe foreign cultivar using a chromosome fragment-substituted line inwhich only a part of a chromosome of an original cultivar is substitutedwith a chromosome fragment derived from a foreign cultivar, the methodcomprising: designing NA markers M1 to M5 so that, for every one targetregion, a DNA marker M2 is defined at an end on an upstream side of thetarget region, or upstream thereof, a DNA marker M1 is defined upstreamof the DNA marker M2, a DNA marker M4 is defined at an end on adownstream side of the target region, or downstream thereof, a DNAmarker M5 is defined downstream of the DNA marker M4, and a DNA markerM2 is defined in the target region; and designing a genome so that asubstitution region, containing the target region, in a chromosome ofthe original cultivar to be substituted with a chromosome fragmentderived from the foreign cultivar is such that an end on an upstreamside of it is between the DNA marker M1 and the DNA marker M2, and anend on a downstream side of the substitution region is between the DNAmarker M4 and the DNA marker M5.(2) A method of creating a new cultivar of the present invention is amethod of creating a new cultivar using a chromosomefragment-substituted line in which only a part of a chromosome of anoriginal cultivar is substituted with a chromosome fragment derived froma foreign cultivar, comprises: (1-1) a step of defining a DNA marker M2at an end on an upstream side of a target region of a chromosome of theoriginal cultivar, or upstream thereof, a step of defining a DNA markerM1 upstream of the DNA marker M2, a step of defining a DNA marker M4 atan end on a downstream side of the target region, or downstream thereof,a step of defining a DNA marker M5 downstream of the DNA marker M4, astep of defining a DNA marker M3 in the target region; (1-2) a step ofmating the chromosome fragment-substituted line and the originalcultivar to obtain a progeny individual in which the DNA marker M3 is ahetero-chromosome region of an allele derived from the original cultivarand an allele derived from a foreign cultivar; (1-3) a step ofself-mating the progeny individual obtained in the step (1-2) to obtaina progeny individual; (1-4) a step of selecting a progeny individual inwhich the DNA marker M1 is a homo-chromosome region of an allele derivedfrom the original cultivar, and the DNA marker M2 and the DNA marker M3is a hetero-chromosome region of an allele derived from the originalcultivar and an allele derived from the foreign cultivar, from a progenyindividual obtained by backcrossing the progeny individual obtained inthe step (1-3), or the progeny individual obtained in the step (1-3);(1-5) a step of self-mating the progeny individual selected in the step(1-4) to obtain a progeny individual; (1-6) a step of selecting aprogeny individual in which the DNA marker M1 and the DNA marker M5 is ahomo-chromosome region of an allele derived from the original cultivar,and the DNA marker M2, the DNA marker M3, and the DNA marker M4 are ahomo-chromosome region of an allele derived from the foreign cultivar,from the progeny individual obtained in the step (1-5), or a progenyindividual obtained by the self-mating the progeny individual obtainedin the step (1-5), wherein the steps (1-1) to (1-6) are performed forthe every one target region, on one or a plurality of the target regionsin the chromosome of the original cultivar.(3) A method of creating a new cultivar of the present invention is amethod of creating a new cultivar using a chromosomefragment-substituted line in which only a part of a chromosome of anoriginal cultivar is substituted with a chromosome fragment derived froma foreign cultivar, the method comprising: (2-1) a step of defining aDNA marker M2 at an end on an upstream side of a target region of achromosome of the original cultivar, a step of defining a DNA marker M1upstream of the DNA marker M2, a step of defining a DNA marker M4 at anend on a downstream side of the target region, or downstream thereof, astep of defining a DNA marker M5 downstream of the DNA marker M4, a stepof defining a DNA marker M3 in the target region; (2-2) a step of matingthe chromosome fragment-substituted line and the original cultivar toobtain a progeny individual in which the DNA marker M3 is ahetero-chromosome region of an allele derived from the original cultivarand an allele derived from the foreign cultivar; (2-3) a step ofself-mating the progeny individual obtained in the step (2-2) to obtaina progeny individual; (2-4) a step of selecting a progeny individual inwhich the DNA marker M5 is a homo-chromosome region of an allele derivedfrom the original cultivar, and the DNA marker M3 and the DNA marker M4are a hetero-chromosome region of an allele derived from the originalcultivar and an allele derived from the foreign cultivar, from theprogeny individual obtained in the step (2-3), or a progeny individualobtained by backcrossing the progeny individual obtained in the step(2-3); (2-5) a step of self-mating the progeny individual selected inthe step (2-4) to obtain a progeny individual; (2-6) a step of selectinga progeny individual in which the DNA marker M1 and the DNA marker M5are a homo-chromosome region of an allele derived from the originalcultivar, and the DNA marker M2, the DNA marker M3, and the DNA markerM4 are a homo-chromosome region of an allele derived from the foreigncultivar, from the progeny individual obtained in the step (2-5), or aprogeny individual obtained by the self-mating the progeny individualobtained in the step (2-5), wherein the steps (2-1) to (2-6) areperformed for the every one target region on one or a plurality of thetarget regions in the chromosome of the original cultivar.(4) A method of creating a new cultivar of the present invention is amethod of creating a new cultivar using a chromosomefragment-substituted line in which only a part of a chromosome of anoriginal cultivar is substituted with a chromosome fragment derived froma foreign cultivar, the method comprising (3-1) a step of defining a DNAmarker M2 at an end on an upstream side of a target region of achromosome of the original cultivar, a step of defining a DNA marker M1upstream of the DNA marker M2, a step of defining at an end on adownstream side of the target region, or downstream thereof, a step ofdefining a DNA marker M5 downstream of the DNA marker M4, a step ofdefining a DNA marker M3 in the target region; (3-2) a step of matingthe chromosome fragment-substituted line, and the original cultivar toobtain a progeny individual in which the DNA marker M3 is ahetero-chromosome region of an allele derived from the original cultivarand an allele derived from the foreign cultivar; (3-3) a step ofself-mating the progeny individual obtained in the step (3-2) to obtaina progeny individual; (3-4) a step of selecting a progeny individual inwhich any one of the DNA marker M1 and the DNA marker M5 is ahomo-chromosome region of an allele derived from the original cultivar,and the other is a hetero-chromosome region of an allele derived fromthe original cultivar and an allele derived from the foreign cultivar,from the progeny individual obtained in the step (3-3), or a progenyindividual obtained by backcrossing the progeny individual obtained inthe step (3-3); (3-5) a step of self-mating the progeny individualselected in the step (3-4) to obtain a progeny individual; (3-6) a stepof selecting a progeny individual in which the DNA marker M1 and the DNAmarker M5 are a homo-chromosome region of an allele derived from theoriginal cultivar, and the DNA marker M2, the DNA marker M3, and the DNAmarker M4 are a homo-chromosome region of an allele derived from theforeign cultivar, from the progeny individual obtained in the step(3-5), or a progeny individual obtained by self-mating the progenyindividual obtained in the step (3-5), wherein the steps (3-1) to (3-6)are performed for the every one target region on one or a plurality ofthe target regions in the chromosome of the original cultivar.(5) The method of creating a new cultivar according to (4) may be suchthat, after the step (3-4) and before the step (3-5), (3-7-1) a step ofobtaining a progeny individual by self-mating the progeny individualobtained in the step (3-4), and (3-7-2) a step of selecting (ii-1) aprogeny individual in which the DNA marker M1 is a homo-chromosomeregion of an allele derived from the original cultivar, and the DNAmarker M2 and the DNA marker M3 are a hetero-chromosome region of anallele derived from the original cultivar and an allele derived from theforeign cultivar, or (ii-2) a progeny individual in which the DNA markerM5 is a homo-chromosome region of an allele derived from the originalcultivar, and the DNA marker M3 and the DNA marker M4 are ahetero-chromosome region of an allele derived from the original cultivarand an allele derived from the foreign cultivar, from the progenyindividual obtained in the step (3-7-1), a progeny individual obtainedby backcrossing the progeny individual obtained in the step (3-7-1), ora progeny individual obtained by backcrossing a progeny individualobtained by self-mating the progeny individual obtained in the step(3-7-1) are performed; the step (3-5) is (3-5′) a step of obtaining aprogeny individual by self-mating the progeny individual selected in thestep (3-7-2); the step (3-6) is (3-6′) a step of selecting a progenyindividual in which the DNA marker M1 and the DNA marker M5 are ahomo-chromosome region of an allele derived from the original cultivar,and the DNA marker M2, the DNA marker M3, and the DNA marker M4 are ahomo-chromosome region of an allele derived from the foreign cultivar,from the progeny individual obtained in the step (3-5), or a progenyindividual obtained by self-mating the progeny individual obtained inthe step (3-5′).(6) The method of creating a new cultivar according to any one of (2) to(4) may be such that the DNA marker M2 is defined at an end on anupstream side of the target region, or in vicinity thereof; the DNAmarker M1 is defined in vicinity of the DNA marker M2; the DNA marker M4is defined at an end on a downstream side of the target region, or invicinity thereof; the DNA marker M5 is defined in vicinity of the DNAmarker M4.(7) The method of creating a new cultivar according to any one of (2) to(4) may be such that the target region is one gene region.(8) The method of creating a new cultivar according to any one of (2) to(4) may be such that the target region is 2 or more gene regions.(9) The method of creating a new cultivar according to any one of (2) to(4) may be such that the original cultivar is an autogamous plant or aself-fertilizing plant.(10) The method of creating a new cultivar according to any one of (2)to (4) may be such that the original cultivar is a cultivar of a Poaceaeplant.(11) The method of creating a new cultivar according to any one of (2)to (4) may be such that the original cultivar is a rice cultivar.(12) The method of creating a new cultivar of according to (11) may besuch that the rice cultivar is Koshihikari.(13) A cultivar of the present invention is a cultivar created using themethod of creating a new cultivar according to any one of (2) to (4),and a target region in a chromosome of the original cultivar issubstituted with a homo-chromosome fragment derived from the foreigncultivar.(14) A progeny individual of the present invention is obtained by matingtwo individuals selected from the group consisting of an individual of acultivar according to (13) and a progeny individual of the individual ofa cultivar according to (13).(15) The progeny individual according to (14) may be such that aplurality of the target regions in a chromosome of the original cultivarare substituted with a homo-chromosome fragment derived from the foreigncultivar.(16) The progeny individual according to (14) may be such thatrespective target regions of the two individuals are different.(17) A progeny individual of the present invention is obtained by using,as a seed parent or a pollen parent, an individual selected from thegroup consisting of the individual of a cultivar according to (13) and aprogeny individual of the individual of a cultivar according to (13),and mating the seed parent or the pollen parent.(18) A method of creating a new cultivar of the present invention has(4-1) a step of using the cultivar according to (13) or the progenyindividual according to (15) as a seed parent, and the cultivaraccording to (13) or the progeny individual according to (15) in whichthe target region is different from that of the seed parent as a pollenparent, and mating the seed parent and the pollen parent to obtain aprogeny individual; (4-2) a step of obtaining a progeny individual byself-mating the progeny individual obtained in the step (4-1); (4-3) astep of selecting a progeny individual in which, in a chromosome of theoriginal cultivar, both of the target region possessed by the seedparent and the target region possessed by the pollen parent aresubstituted with a homo-chromosome fragment derived from the foreigncultivar, from the progeny individual obtained in the step (4-2).(19) The method of creating a new cultivar according to (18) may furtherhave, after the step (4-3), (4-4) a step of selecting two individuals inwhich the target regions are different as a seed parent and a pollenparent, from the group consisting of the cultivar according to (13) or aprogeny individual according to (15), and the individual selected in thestep (4-3), and mating them to obtain a progeny individual; (4-5) a stepof obtaining a progeny individual by self-mating the progeny individualobtained in the step (4-4); (4-6) a step of selecting a progenyindividual in which, in a chromosome of the original cultivar, both ofthe target region possessed by the seed parent and the target regionpossessed by the pollen parent are substituted with a homo-chromosomefragment derived from the foreign cultivar, from the progeny individualobtained in the step (4-5); (4-7) a step of repeating the steps (4-4) to(4-6) once or more.(20) A method of discriminating a plant cultivar of the presentinvention is a method of discriminating whether or not a plantindividual is a particular cultivar created using the method of creatinga new cultivar according to any one of (2) to (4), the methodcomprising: typing one or more of the DNA markers selected from thegroup consisting of the DNA markers M1 to M5 by genome analysis of theplant individual; and discriminating that the plant individual is theparticular cultivar when the resulting typing result is consistent withthe result of the particular cultivar.(21) A new cultivar of the present invention is a progeny cultivar of achromosome fragment-substituted line in which a part of a chromosome issubstituted with a chromosome fragment derived from the foreigncultivar; one or a plurality of target regions of a chromosome regionsare substituted with a chromosome fragment derived from the foreigncultivar; a length of the chromosome fragment is controlled by a DNAmarker defined upstream of the target region, and a DNA marker defineddownstream of a target region.(22) Koshihikari of the present invention is Oryza sativa L. cultivarKoshihikari kazusa 4go, which has an accession number of internationaldepository of FERM ABP-11140.(23) A progeny individual of the present invention is obtained by matingtwo individuals selected from the group consisting of the individual ofa cultivar according to (22) and a progeny individual according to (17).(24) A method of discriminating a cultivar of the present invention is amethod of discriminating whether or not a plant individual is aparticular cultivar, the method comprising: letting SP-4009 to be a DNAmarker M1, letting G2003 to be a DNA marker M2, letting G2002 to be aDNA marker M3, letting SP-462 to be a DNA marker M4, and letting SP-1259to be a DNA marker M5; typing one or more of the DNA markers selectedfrom the group consisting of the DNA markers M1 to M5 by genome analysisof the plant individual; and discriminating that the plant individual isOryza sativa L. cultivar Koshihikari eichi 4go or Oryza sativa L.cultivar Koshihikari kazusa 4go when the resulting typing result isconsistent with the result of Oryza sativa L. cultivar Koshihikari eichi4go or Oryza sativa L. cultivar Koshihikari kazusa 4go.(25) A method of discriminating a cultivar of the present invention is amethod of discriminating whether or not a plant individual is aparticular cultivar, the method comprising: letting SP-2032 to be a DNAmarker M1, letting SP-170 to be a DNA marker M2, letting SP-4028 to be aDNA marker M3, letting SP4038 to be a DNA marker M4, and letting SP-4030to be a DNA marker M5; typing one or more of the DNA markers selectedfrom the group consisting of the DNA markers M1 to M5 by genome analysisof the plant individual; and discriminating that the plant individual isOryza sativa L. cultivar Koshihikari eichi 2go or Oryza sativa L.cultivar Koshihikari kazusa 4go when the resulting typing result isconsistent with the result of Oryza sativa L. cultivar Koshihikari eichi2go or Oryza sativa L. cultivar Koshihikari kazusa 4go.(26) A method of discriminating a cultivar of the present invention isthe method of discriminating whether or not a plant individual is aparticular cultivar, the method comprising: letting P-2513 to be a DNAmarker M1, letting SP-586 to be a DNA marker M2, letting SP-2254 to be aDNA marker M3, letting SP-1603 to be a DNA marker M4, and letting SP-604to be a DNA marker M5; typing one or more of the DNA markers selectedfrom the group consisting of the DNA markers M1 to M5 by genome analysisof the plant individual; and discriminating that the plant individual isOryza sativa L. cultivar Koshihikari eichi 3go or Oryza sativa L.cultivar Koshihikari kazusa 4go when the resulting typing result isconsistent with the result of Oryza sativa L. cultivar Koshihikari eichi3go or Oryza sativa L. cultivar Koshihikari kazusa 4go.

Effects of the Invention

By using the method of plant genome design of the present invention, andthe method of creating a new cultivar of the present invention usingthis genome design method, a region of a chromosome of an originalcultivar to be substituted with a homo-chromosome fragment derived froma foreign cultivar can be controlled. For this reason, an objectivecharacter can be introduced into an original cultivar while apossibility that many other genes having the unknown function other thanan objective character gene are introduced into a chromosome of anoriginal cultivar, and a possibility that a preferable characterpossessed by an original cultivar is damaged are kept to a minimum.

In addition, by using a new cultivar created by the method of creating anew cultivar of the present invention or a progeny individual thereof asa parent individual and thereby creating a new cultivar, it can beobtained a progeny individual in which all homo-chromosome fragmentsderived from foreign cultivars possessed by a seed parent and a pollenparent, respectively, are accumulated. As a result, plural kinds ofcharacters of an original cultivar can be simply and safely improved,while a possibility that a preferable character possessed by an originalcultivar is damaged is kept to a minimum.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing a target region T on a chromosome G of anoriginal cultivar, a chromosome fragment G derived from a foreigncultivar to be introduced into this chromosome G, and DNA markers M1 toM5.

FIG. 2A is a view showing a chromosome region of a progeny individualpreferable in a step (1-4), among progeny individuals obtained step(1-3) in a first method of creating a new cultivar of the presentinvention. In the figure, a non-filled bold line shows an allele derivedfrom an original cultivar, and a filled bold line shows an allelederived from a foreign cultivar, respectively.

FIG. 2B is a view showing a chromosome region of a progeny individualpreferable in a step (1-4), among progeny individuals obtained in a step(1-3) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 2C is a view showing a chromosome region of a progeny individualpreferable in a step (1-4), among progeny individuals obtained in a step(1-3) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 2D is a view showing a chromosome region of a progeny individualpreferable in a step (1-6), among progeny individuals obtained in a step(1-5) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 2E is a view showing a chromosome region of a progeny individualpreferable in a step (1-6), among progeny individuals obtained in a step(1-5) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 2F is a view showing a chromosome region of a progeny individualpreferable in a step (1-6), among progeny individuals obtained in a step(1-5) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 3A is a view showing a chromosome region of a progeny individualpreferable in a step (2-4), among progeny individuals obtained in a step(2-3) of a second method of creating a new cultivar of presentinvention. In the figure, a non-filled bold line shows an allele derivedfrom an original cultivar, and a filled bold line shows an allelederived from a foreign cultivar, respectively.

FIG. 3B is a view showing a chromosome region of a progeny individualpreferable in a step (2-4), among progeny individuals obtained in a step(2-3) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 3C is a view showing a chromosome region of a progeny individualpreferable in a step (2-4), among progeny individuals obtained in a step(2-3) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 3D is a view showing a chromosome region of a progeny individualpreferably in a step (2-6), among progeny individuals obtained in a step(2-5) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 3E is a view showing a chromosome region of a progeny individualpreferable in a step (2-6), among progeny individuals obtained in a step(2-5) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 3F is a view showing a chromosome region of a progeny individualpreferable in a step (2-6), among progeny individuals obtained in a step(2-5) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 4A is a view showing a chromosome region of a progeny individualpreferable in a step (3-4), among progeny individuals obtained in a step(3-3) in a third method of creating a new cultivar of the presentinvention. In the figure, a non-filled bold line shows an allele derivedfrom an original cultivar, and a filled bold line shows an allelederived from a foreign cultivar, respectively.

FIG. 4B is a view showing a chromosome region of a progeny individualpreferable in a step (3-4), among progeny individuals obtained in a step(3-3) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 4C is a view showing a chromosome region of a progeny individualpreferable in a step (3-4), among progeny individuals obtained in a step(3-3) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 4D is a view showing a chromosome region of a progeny individualpreferable in a step (3-4), among progeny individuals obtained in a step(3-3) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 4E is a view showing a chromosome region of a progeny individualpreferable in a step (3-4), among progeny individuals obtained in a step(3-3) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 4F is a view showing a chromosome region of a progeny individualpreferable in a step (3-4), among progeny individuals obtained in a step(3-3) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 5A is a view showing a chromosome region of a progeny individualpreferable in a step (3-6), among progeny individuals obtained in a step(3-5) in a third method of creating a new cultivar of the presentinvention. In the figure, a non-filled bold line shows an allele derivedfrom an original cultivar, and a filled bold line shows an allelederived from a foreign cultivar, respectively.

FIG. 5B is a view showing a chromosome region of a progeny individualpreferable in a step (3-6), among progeny individuals obtained in a step(3-5) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 5C is a view showing a chromosome region of a progeny individualpreferable in a step (3-6), among progeny individuals obtained in a step(3-5) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 5D is a view showing a chromosome region of a progeny individualpreferable in a step (3-6), among progeny individuals obtained in a step(3-5) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 5E is a view showing a chromosome region of a progeny individualpreferable in a step (3-6), among progeny individuals obtained in a step(3-5) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 5F is a view showing a chromosome region of a progeny individualpreferable in a step (3-6), among progeny individuals obtained in a step(3-5) in the same method. In the figure, a non-filled bold line shows anallele derived from an original cultivar, and a filled bold line showsan allele derived from a foreign cultivar, respectively.

FIG. 6A is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 6B is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 6C is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 6D is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 7A is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 7B is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 7C is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 7D is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 7E is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 7F is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 7G is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 8A is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 8B is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 8C is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 8D is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 9A is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 9B is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 9C is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 9D is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 9E is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 9F is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 9G is a view showing a chromosome region of a progeny individualrelatively preferable in a step (3-7-2), among progeny individualsobtained in a step (3-7-1). In the figure, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively.

FIG. 10A is a schematic view showing a method of creating a cultivar inwhich three target regions (target regions A, B, C) in a chromosome ofan original cultivar are substituted with a chromosome fragment derivedfrom a foreign cultivar.

FIG. 10B is a schematic view showing a method of creating a cultivar inwhich three target regions (target regions A, B, C) in a chromosome ofan original cultivar are substituted with a chromosome fragment derivedfrom a foreign cultivar.

FIG. 11A is a schematic view showing a method of creating a cultivar inwhich four target regions (target regions A, B, C, D) in a chromosome ofan original cultivar are substituted with a chromosome fragment derivedfrom a foreign cultivar.

FIG. 11B is a schematic view showing a method of creating a cultivar inwhich four target regions (target regions A, B, C D) in a chromosome ofan original cultivar are substituted with a chromosome fragment derivedfrom a foreign cultivar.

FIG. 11C is a schematic view showing a method of creating a cultivar inwhich four target regions (target regions A, B, C, D) in a chromosome ofan original cultivar are substituted with a chromosome fragment derivedfrom a foreign cultivar.

FIG. 12A is a schematic view showing a method of creating a cultivar inwhich five target regions (target regions A, B, C, D, E) in a chromosomeof an original cultivar are substituted with a chromosome fragmentderived from a foreign cultivar.

FIG. 12B is a schematic view showing a method of creating a cultivar inwhich five target regions (target regions A, B, C, D, E) in a chromosomeof an original cultivar are substituted with a chromosome fragmentderived from a foreign cultivar.

FIG. 12C is a schematic view showing a method of creating a cultivar inwhich five target regions (target regions A, B, C, D, E) in a chromosomeof an original cultivar are substituted with a chromosome fragmentderived from a foreign cultivar.

FIG. 12D is a schematic view showing a method of creating a cultivar inwhich five target regions (target regions A, B, C, D, E) in a chromosomeof an original cultivar are substituted with a chromosome fragmentderived from a foreign cultivar.

FIG. 13A is a schematic view showing a method of creating a cultivar inwhich six target regions (target regions A, B, C, D, E, F) in achromosome of an original cultivar are substituted with a chromosomefragment derived from a foreign cultivar.

FIG. 13B is a schematic view showing a method of creating a cultivar inwhich six target regions (target regions A, B, C, D, E, F) in achromosome of an original cultivar are substituted with a chromosomefragment derived from a foreign cultivar.

FIG. 13C is a schematic view showing a method of creating a cultivar inwhich six target regions (target regions A, B, C, D, E, F) in achromosome of an original cultivar are substituted with a chromosomefragment derived from a foreign cultivar.

FIG. 13D is a schematic view showing a method of creating a cultivar inwhich six target regions (target regions A, B, C, D, E, F) of achromosome in an original cultivar are substituted with a chromosomefragment derived from a foreign cultivar.

FIG. 13E is a schematic view showing a method of creating a cultivar inwhich six target regions (target regions A, B, C, D, E, F) in achromosome of an original cultivar are substituted with a chromosomefragment derived from a foreign cultivar.

FIG. 14A is a schematic view showing a method of creating a cultivar P6(ABCDEF), as an existing probability that an objective progenyindividual is present in a one time selected population is 1/64 to 1/16.

FIG. 14B is a schematic view showing a method of creating a cultivar P6(ABCDEF), as an existing probability that an objective progenyindividual is present in a one time selected population is 1/64 to 1/16.

FIG. 14C is a schematic view showing a method of creating a cultivar P6(ABCDEF), as an existing probability that an objective progenyindividual is present in a one time selected population is 1/64 to 1/16.

FIG. 15 is a view showing a DNA marker used in creating Koshihikarieichi 4go.

FIG. 16 is a view schematically showing a genome of Koshihikari eichi4go.

FIG. 17 is a view comparing lodging resistance of Koshihikari eichi 4goand Koshihikari. A front agricultural field is the field of Koshihikari,and back agricultural field is the field of Koshihikari eichi 4go.

FIG. 18 is a view showing a DNA marker used in creating Koshihikarieichi 2go.

FIG. 19 is a view schematically showing a genome of Koshihikari eichi2go.

FIG. 20 is a view showing a DNA marker used in creating Koshihikarieichi 3go.

FIG. 21 is a view schematically showing a genome of Koshihikari eichi3go.

FIG. 22 is a view schematically showing a genome of Koshihikari kazusa4go.

CARRYING OUT THE INVENTION

In the present invention, the chromosome fragment-substituted line meansa line in which only a part of a chromosome of an original cultivar issubstituted with a chromosome fragment derived from a foreign cultivar.

The foreign cultivar is not particularly limited as far as it is acultivar other than an original cultivar, and may be a cultivar of aplant which is the same species as that of an original cultivar, may bea cultivar of a plant which is a different species from that of anoriginal cultivar, and may be a cultivar other than a plant such as ananimal and the like.

In the present invention, the cultivar means a population which is thesame species of a plant, but can be clearly discriminated from otherspecies in the same species in a certain character, due to differentgenetic construction.

In the present invention, the target region means a region in achromosome of an original cultivar, which is aimed at being substitutedwith a chromosome fragment derived from a foreign cultivar. For example,in a cultivar of a plant for which genetic information has beensufficiently elucidated, such as rice, wheat, and Arabidopsis, a newcultivar improved in a character of an original cultivar can be createdby substituting a particular chromosome region containing an objectivecharacter gene with a chromosome fragment derived from a foreigncultivar. Herein, the target region of an original cultivar is notparticularly limited as far as it is a region corresponding to a regionof a part substituted with a chromosome fragment derived from a foreigncultivar among a chromosome of a chromosome fragment-substituted lineused as a parent individual, and the target region of the originalcultivar may be one gene region, or may be a region containing two ormore genes. For example, when a foreign cultivar is different speciesfrom that of an original cultivar, it is preferable that a region is onegene region. In addition, when a foreign cultivar is a related speciesof an original cultivar, such as when a foreign cultivar is othercultivar which is the same species of an original cultivar, the targetregion may be one gene region, or may be a region containing two or moregenes.

This gene region may be only a translation region, or may be a regioncontaining a non-translation region such as intron, a control regionsuch as a promoter region and a terminator region in addition to thetranslation region.

The DNA markers in the present invention are not particularly limited asfar as it can discriminate a chromosome derived from an originalcultivar and a chromosome derived from a foreign cultivar, that is, itcan detect a difference in a DNA sequence on a chromosome between theoriginal cultivar and the foreign cultivar, and a DNA marker which isnormally used in the gene analysis field can be used. These DNA markersmay be, for example, a RFLP (Restriction Fragment Length Polymorphism)marker or a marker which can detect gene polymorphism such as SNP(Single Nucleotide Polymorphism), and can detect a difference in therepetition number of SSR (Simple Sequence Repeats).

Discrimination of an allele derived from the original cultivar and anallele derived from the foreign cultivar using these DNA markers can beperformed by a conventional method. For example, PCR is performed asfollows: employing a DNA extracted from each individual as a template;and using primers which can specifically hybridize with particular SNPand SSR. Then, by detecting the presence or the absence of the PCRproduct using an electrophoresis method or the like, each polymorphismof SNP and SSR can be discriminated between the original cultivar andthe foreign cultivar. Alternatively, by detecting a pattern of a DNAfragment using an electrophoresis method or the like after a DNAextracted from each individual is treated with a restriction enzyme,each polymorphism can be discriminated similarly. Primers which canhybridize specifically with particular SNP and SSR can be designed by aconventional method using a primer design tool which is generally used,depending on a nucleotide sequence of SNP and SSR. In addition, designedprimers can be synthesized using any of methods which are well-known inthe present technical field.

As these DNA markers, the known DNA markers can be appropriately used.Alternatively, newly prepared DNA markers may be used. For example, whenthe known DNA markers regarding rice are used, SNP markers disclosed inPatent document 2, and DNA markers published in Rice Genome ResearchProgram (http://rgp.dna.affrc.go.jp/publicdata.html) can be used. Whenthe known DNA markers regarding barley are used, DNA markers publishedin GrainGenes: A Database for Triticeae and Avena(http://wheat.pw.usda.gov/GG2/index.shtml), CR-EST:The IPK Crop ESTDatabase (http://pgrc.ipk-gatersleben.de/est/index.php) can be used.When the known DNA markers regarding sorghum are used, DNA markerspublished in GRAMENE (http://www.gramene.org/db/markers/marker view) canbe used. When the known DNA markers regarding wheat are used, DNAmarkers published in GrainGenes: A Database for Triticeae and Avena,WHEAT CAP (http://maswheat.ucdavis.edu/) can be used. When the known DNAmarkers regarding corn are used, DNA markers published in MaizeGDB(http://www.maizegdb.org/) can be used. In addition, DNA markers ofother serials are disclosed in GRAMENE, and these can be also used.

First, the method of plant genome design of the present invention isexplained.

The method of plant genome design of the present invention is a methodof designing a genome of a plant in which one or plural target regionsin a chromosome of an original cultivar is substituted with a chromosomefragment derived from a foreign cultivar, using a chromosomefragment-substituted line in which only a part of a chromosome of theoriginal cultivar is substituted with a chromosome fragment derived fromthe foreign cultivar. In this method, DNA markers M1 to M5 satisfyingthe following requirement (i) are defined for every one target region.That is, in a substitution region in a chromosome of the originalcultivar which contains this target region and is to be substituted witha chromosome fragment derived from the foreign cultivar, a genome isdesigned so that an end on an upstream side thereof is between DNAmarkers M1 and M2, and an end on a downstream side thereof is betweenDNA markers M4 and M5.

(i) The DNA marker M2 is defined at an end on an upstream of the targetregion, or upstream thereof. The DNA marker M1 is defined upstream ofthe DNA marker M2. The DNA marker M4 is defined at an end on adownstream side of the target region, or downstream thereof. The DNAmarker M5 is defined downstream of the DNA marker M4. The DNA marker M3is defined in the target region.

In the present invention, the upstream side means a short arm side of achromosome, and the downstream means a long arm side of a chromosome.

By defining respective DNA markers M1 to M5 so as to satisfy therequirement (i), a length of a chromosome fragment derived from aforeign cultivar to be introduced into a chromosome of the originalcultivar, that is, a substitution region in a chromosome of the originalcultivar to be substituted with this chromosome fragment can becontrolled. For this reason, by using the method of plant genome designof the present invention, a genome can be designed so that a genederived from the foreign cultivar of a target is introduced into achromosome of the original cultivar, while a probability that many othergenes other than an objective gene are introduced into a chromosome ofthe original cultivar and a possibility that a gene other than anobjective gene present in vicinity of a target region is substitutedwith a chromosome of the original cultivar derived from the foreigncultivar are kept to a minimum.

Respective DNA markers M1 to M5 can be designed based on the knowngenetic information of a plant species to which each cultivar belongs.Genetic information of each cultivar is available, for example, in NCBI(National center for Biotechnology Information) and DDBJ (DNA Data Bankof Japan) which are international nucleotide sequence database.Particularly, genetic information of each cultivar of rice is availablein KOME (Knowledge-based Oryza Molecular biological Encyclopedia,http://cdna01.dna.affrc.go.jp/cDNA/).

FIG. 1 is a view showing a target region T on a chromosome G of anoriginal cultivar, a foreign cultivar-derived chromosome fragment L tobe substituted, and DNA markers M1 to M5. An end on an upstream side ofthe foreign cultivar-derived chromosome fragment L, that is, an end onan upstream side of a substituent region in a chromosome of an originalcultivar, which is substituted with the foreign cultivar-derivedchromosome fragment L, is between the DNA markers M1 and M2. On theother hand, an end on a downstream side of the foreign cultivar-derivedchromosome fragment L, that is, an end on a downstream side of asubstitution region in a chromosome of an original cultivar, which issubstituted with the foreign cultivar-derived chromosome fragment L, isbetween the DNA markers M4 and M5. For this reason, letting a distancebetween the DNA markers M1 and M2 to be d1, letting distance between theDNA markers M2 and M4 to be d2, and letting a distance between the DNAmarkers M4 and M5 to be d3, a length of the foreign cultivar-derivedchromosome fragment L (length of substitution region) is expressed byfollowing equation (1).

d2≦length of foreign cultivar-derived chromosome fragmentL≦d1+d2+d3  Equation (1)

By setting the DNA marker M2 on an upstream side (a direction away fromthe target region T) of the chromosome G of the original cultivar, alength of the foreign cultivar-derived chromosome fragment L becomeslong. On the other hand, by setting the DNA marker M2 on a downstreamside (a direction approaching a target region T) of the chromosome G ofthe original cultivar, a length of the foreign cultivar-derivedchromosome fragment L becomes short. Similarly, by setting the DNAmarker M4 on a downstream side of the chromosome G of the originalcultivar, a length of the foreign cultivar-derived chromosome fragment Lbecomes long and, by setting on an upstream side of the chromosome G ofthe original cultivar, a length of the foreign cultivar-derivedchromosome fragment L becomes short.

In addition, when a distance d1 between the DNA markers M1 and M2 islong, a range in which an end on an upstream side of the foreigncultivar-derived chromosome fragment L can exist is widened. For thisreason, it becomes difficult to define a length of the foreigncultivar-derived chromosome fragment L to be introduced. On the otherhand, when this distance d1 is short, a range in which an end on anupstream side of the foreign cultivar-derived chromosome fragment L canexist is narrowed. For this reason, it becomes easy to define a lengthof the foreign cultivar-derived chromosome fragment L to be introduced.

Similarly, when a distance d3 between the DNA markers M4 and M5 is long,a range in which an end on a downstream side of the foreigncultivar-derived chromosome fragment L can exist is widened, and itbecomes difficult to define the length of the foreign cultivar-derivedchromosome fragment L to be introduced. When this distance d3 is short,a range in which an end on a downstream side of the foreigncultivar-derived chromosome fragment L can exist is narrowed, it becomeeasy to define a length of the foreign cultivar-derived chromosomefragment L to be introduced.

As a length of a foreign cultivar-derived chromosome fragment L becomeslonger, a possibility that genes presented on both sides of the targetregion of T are introduced into the original cultivar together with anobjective gene presented in the target region T becomes higher.Introduction of a gene other than an objective gene into a chromosome ofthe original cultivar results in that a gene other than an objectivegene presented in the original cultivar is substituted with the foreigncultivar-derived chromosome fragment L. As a result, there is apossibility that an excellent character possessed by the originalcultivar is carelessly damaged. As a gene other than an objective geneto be introduced into a chromosome of the original cultivar is fewer,that is, as a length of the foreign cultivar-derived chromosome fragmentL is closer to a length of the target region of T, a possibility that anexcellent character of the original cultivar is substituted can besuppressed. Therefore, it is preferable that a length of the foreigncultivar-derived chromosome fragment L is closer to a length of thetarget region of T.

As the DNA markers M2 and M1 are closer to an end of an upper streamside of the target region T, and as the DNA markers M4 and M5 are closerto an end on a downstream side of the target region T, a length offoreign cultivar-derived chromosome fragment L becomes shorter. As aresult, a chromosome region other than the target region T of theforeign cultivar-derived chromosome fragment L to be introduced into achromosome of the original cultivar can be shorter. Then, it ispreferable that the DNA marker M2 is defined in vicinity of an end of anupstream side of the target region T, and it is more preferable that theDNA marker M2 is on the same site as that of an end on an upstream sideof the target region T. In addition, it is preferable that the DNAmarker M1 is defined in vicinity on an upstream side of the DNA markerM2. On the other hand, it is preferable that the DNA marker M4 isdefined in vicinity of an end on a downstream side of the target regionof the T, and it is more preferable that the DNA marker M4 is on thesame site as that of an end on a downstream side of the target region T.In addition, it is preferable that the DNA marker M5 is defined invicinity on a downstream side of the DNA marker M4.

However, when a distance d1 between the DNA markers M1 and M2, adistance d2 between the DNA markers M2 and M4, and a distance d3 betweenthe DNA markers M4 and M5 become too short, respectively, arecombination frequency of a chromosome becomes small. For this reason,as far as a size of a population from which a progeny individual isselected is not increased, it becomes difficult to obtain an objectiveprogeny individual (a progeny individual in which recombination of achromosome has occurred).

When a foreign cultivar is a related species of the original cultivar,DNA sequences of chromosomes of both cultivars have high homology. Forthis reason, even when a length of the foreign cultivar-derivedchromosome fragment L is long and thereby a gene in vicinity of anobjective gene (target regions T) is substituted with the foreigncultivar derived gene together with the objective gene, there is apossibility that an excellent character of the original cultivar is notdamaged.

From the foregoing, it is preferable that defining of these DNA markersM1, M2, M4, and M5 is appropriately determined in view of a length ofthe target region T, whether the original cultivar and the foreigncultivar are related species or a distant species, and a size of apopulation to be selected.

Currently, although sequence information of a gene has been revealed,many genes whose function is unknown are present. In addition, even in agene whose function is thought to be known, there are still many casesthat the unknown new function is found by analysis thereafter. Inprinciple, it is possible to elucidate the function of such the gene byintroducing a chromosome fragment encoding the gene, whose function isunknown, into a chromosome of the original cultivar which has notinherently this gene and then comparing and studying a biologicalcharacter such as physiological activity possessed by the resultingcultivar with that of the original cultivar. However, in a cultivarimprovement method using the previous procedure such as the MAS methodand the like, it is difficult to strictly control a chromosome fragmentderived from the foreign cultivar to be introduced into the originalcultivar. Therefore, information such as what a gene is encoded inaddition to an objective gene in the introduced chromosome fragment, orwhat a gene was encoded in a chromosome region of the original cultivarlost by substitution with this chromosome fragment is unknown in manycases. For this reason, in the case of a cultivar having a genomedesigned so as to introduce a foreign cultivar-derived chromosomefragment by the previous method, it is very difficult to preciselyassess whether a biological character different from that of theoriginal cultivar is the function expressed by an introduced objectivegene or not. In addition, even in the case where an objective charactercould be introduced, when other character has been also varied, it isdifficult to determine that whether this variation is due to the unknownfunction of the introduced objective gene, or due to a gene differentfrom this gene.

To the contrary, in a genome designed by the method of plant genomedesign of the present invention, a length of the foreigncultivar-derived chromosome fragment L, and a substitution region of thechromosome G of the original cultivar to be substituted with thisforeign cultivar-derived chromosome fragment L can be more strictlydefined than previously. For this reason, in the case of a cultivarhaving a genome designed so as to introduce the foreign cultivar-derivedchromosome fragment L using the method of plant genome design of thepresent invention, it becomes possible to assess a character introducedinto the original cultivar by this foreign cultivar-derived chromosomefragment L at a higher precision than previously. Therefore, the methodof plant genome design of the present invention can be suitably usedalso in analysis of the gene function.

Then, the method of creating a new cultivar of a present invention willbe explained. The method of creating a new cultivar of the presentinvention utilizes the method of plant genome design of the presentinvention. Specifically, there are following four kinds (first tofourth) of production methods.

In the method of creating a new cultivar of the present invention, theoriginal cultivar is not particularly limited as far as it is a plantcultivar, but it is preferable that the original cultivar is such asPoaceae, Fabaceae, Brassicaceae, Rutaceae, Malvaceae, Asteraceae,Amaranthaceae, Euphorbiaceae, Convolvuaceae, or Liliaceae. As a plant ofPoaceae, for example, rice, corn, sorghum bicolor, wheat, barley, rye,Japanese barnyard millet, and sorghum are preferable. In addition, as aplant of Fabaceae, for example, peanut, chick-pea, soybean max, commonbean, bird's-foot trefoil, and medicago are preferable. As a plant ofBrassicacae, for example, thale-cross, oilseed rape, shepherd's-purse,radish, cabbage, and wasabi are preferable. As a plant of Rutaceae, forexample, orange is preferable. As a plant of Malvaceae, for example,cotton is preferable. As a plant of Asteraceae, for example, helianthus,lettuce, Zinnia-elegans, tomato, potato, chili pepper, and tobacco arepreferable. As a plant of Amaranthaceae, for example, sugar beet ispreferable and, as a plant of Euphorbiaceae, for example, Euphorbiaesula, and cassaya are preferable. As a plant of Convolvuaceae, forexample, Japanese morning glory is preferable and, as a plant ofLiliaceae, for example, onion is preferable.

In the method of creating a new cultivar of the present invention, it ispreferable that the chromosome fragment-substituted line is,particularly, a line of an autogamous plant or a self-fertile plantbecause an uncertain element in genome design can be reduced. Herein,self-propagation means mating using a self as a mate. Specifically, inthe case of a hermaphrodite plant, self-propagation is that an ovule isfertilized by autogamy to produce a seed, that is, selfmating.

Particularly, the method of creating a new cultivar of the presentinvention can create a new cultivar relatively safely and stably withoutusing a gene recombinant method. For this reason, the chromosomefragment-substituted line used in the present invention is preferablysuch that an edible plant is the original cultivar, more preferably suchthat rice, wheat, corn, or soybean is the original cultivar, and furtherpreferably such that rice is the original cultivar. A rice cultivar ispreferably Koshihikari, Habataki, or IR64, particularly preferablyKoshihikari.

The chromosome fragment-substituted line used in the present inventionmay be created by the conventional method, or may be available from anorganization such as National Institute of Agrobiological Sciences RiceGenome Resource Center.

In the first method of creating a new cultivar of the present invention,the following steps (1-1) to (1-6) are performed for every one targetregion on one or plural target regions in a chromosome of an originalcultivar, using a chromosome fragment-substituted line in which only apart of a chromosome of an original cultivar is substituted with achromosome fragment derived from a foreign cultivar.

(1-1) A step of defining a DNA marker M2 at an end on an upstream sideof a target region, or upstream thereof; a step of defining a DNA markerM1 upstream of the DNA marker M2; a step of defining a DNA marker M4 atan end on a downstream side of the target region, or downstream thereof;a step of defining a DNA marker M5 downstream of the DNA marker M4; astep of defining a DNA marker M3 in the target region.(1-2) A step of mating a chromosome fragment-substituted line and anoriginal cultivar to obtain a progeny individual in which the DNA markerM3 is a hetero-chromosome region of an allele derived from the originalcultivar and an allele derived from a foreign cultivar.(1-3) A step of self-mating the progeny individual obtained in the step(1-2) to obtain a progeny individual.(1-4) A step of selecting a progeny individual in which the DNA markerM1 is a homo-chromosome region of an allele derived from the originalcultivar, and the DNA markers M2 and M3 are a hetero-chromosome regionof an allele derived from the original cultivar and an allele derivedfrom the foreign cultivar, from the progeny individual obtained in thestep (1-3); or a progeny individual obtained by backcrossing the progenyindividual obtained in the step (1-3).(1-5) A step of self-mating the progeny individual selected in the step(1-4) to obtain a progeny individual.(1-6) A step of selecting a progeny individual in which the DNA markersM1 and M5 are a homo-chromosome region of an allele derived from theoriginal cultivar, and the DNA markers M2, M3 and M4 are ahomo-chromosome region of an allele derived from the foreign cultivarfrom the progeny individual obtained in the step (1-5); or a progenyindividual obtained by self-mating the progeny individual obtained inthe step (1-5).

Each step will be explained below.

First, as the step (1-1), a DNA marker M2 is defined at an end on anupstream side of a target region in a chromosome of an originalcultivar, or upstream thereof, and a DNA marker M1 is defined upstreamof the DNA marker M2. On the other hand, a DNA marker M4 is defined atan end on a downstream side of this target region, or downstreamthereof, and a DNA marker M5 is defined downstream of the DNA marker M4.And, in this target region, a DNA marker M3 is defined. That is,respective DNA markers M1, M2, M4, M5 are defined so that an end on anupstream side of a chromosome fragment derived from a foreign cultivarto be introduced into a chromosome region (region containing a targetregion) of the original cultivar by substitution is between DNA markersM1 and M2, and an end on a downstream side thereof is between the DNAmarkers M4 and M5.

Specifically, defining of respective DNA markers M1 to M5 is the same asthat of the method of plant genome design of the present invention.

By defining the DNA markers M1 to M5 like this, in creating a newcultivar, a length of a chromosome fragment derived from the foreigncultivar to be introduced into a chromosome of the original cultivar canbe controlled. As a result, a gene other than an objective gene can beeffectively suppressed from being introduced into a chromosome of theoriginal cultivar. Further, it becomes possible to effectively suppressa gene other than an objective gene present in vicinity of the targetregion from being substituted with a chromosome of the original cultivarderived from the foreign cultivar.

Then, as the step (1-2), a chromosome fragment-substituted line and theoriginal cultivar are mated to obtain a progeny individual in which theDNA marker M3 is a hetero-chromosome region of an allele derived fromthe original cultivar and an allele derived from the foreign cultivar.The chromosome fragment-substituted line as a seed parent, and theoriginal cultivar as a pollen parent may be mated, or the originalcultivar as a seed parent and the chromosome fragment-substituted lineas a pollen parent may be mated.

Usually, in mating, genes possessed by a parent individual are randomlyarranged in a gamete. For this reason, although a progeny individualselected by the DNA marker has a gene encoding an objective character,how other gene regions are changed from the parent individual isunknown. For this reason, it is difficult to determine that a phenotypiccharacter of the resulting progeny individual is due to a chromosomeregion linked with the DNA marker, or influence of a gene present inother chromosome region.

In the present invention, the chromosome fragment-substituted line andthe original cultivar of this chromosome fragment-substituted line areused as the parent individual. In this chromosome fragment-substitutedline, other chromosome regions other than a chromosome fragment derivedfrom a foreign cultivar all have the same genes as those of the originalcultivar. Therefore, a chromosome region of the resulting progenyindividual is such that chromosome regions other than a chromosomefragment derived from the foreign cultivar all become to have the samegenes as those of the original cultivar. For this reason, in thisprogeny individual, it becomes possible to easily determine theinfluence by the chromosome fragment derived from the foreign cultivar.

In the method of creating a new cultivar of the present invention,mating may be natural mating, but since a seed parent and a pollenparent can be assuredly identified, artificial mating is preferable.Herein, the artificial mating method is not particularly limited as faras it is a method which can make a pistil of a seed parent receive apollen collected from a pollen parent to fertilize it, but can beperformed by the conventional method.

As the step (1-3), the progeny individual obtained in the step (1-2) isself-mated to obtain a progeny individual. Thereafter, as the step(1-4), a progeny individual in which the DNA marker M1 is ahomo-chromosome region of an allele derived from the original cultivar,and the DNA markers M2 and M3 are a hetero-chromosome region of anallele of the original cultivar and an allele derived from the foreigncultivar is selected, from the progeny individual obtained in the step(1-3); or a progeny individual obtained by backcrossing the progenyindividual obtained in the step (1-3).

FIG. 2A to FIG. 2C are views showing a chromosome region of a progenyindividual preferable in the step (1-4) among progeny individualsobtained in the step (1-3). In the figure, a non-filled bold line showsan allele derived from the original cultivar, and a filled bold lineshows an allele derived from the foreign cultivar. First, from theprogeny individual obtained in the step (1-3), a progeny individual (1a)in which the DNA marker M1 is a homo-chromosome region of an allelederived from the original cultivar, and the DNA markers M2 and M3 are ahetero-chromosome region; a progeny individual (1b) in which the DNAmarker M1 is a hetero-chromosome region, and the DNA markers M2 and M3are a homo-chromosome region of an allele derived from the foreigncultivar; a progeny individual (1c) in which the DNA marker M1 is ahomo-chromosome region of an allele derived from the original cultivar,and the DNA markers M2 and M3 are a homo-chromosome region of an allelederived from the foreign cultivar; are selected, respectively. Herein,the progeny individual (1a) is a progeny individual finally selected inthe step (1-4). The progeny individual (1b) and the progeny individual(1c) are further backcrossed with an individual of each originalcultivar, and then the progeny individual (1a) can be selected from theresulting progeny individual.

Then, as the step (1-5), by self-mating the progeny individual (1a)selected in the step (1-4) like this, a progeny individual is obtained.Thereafter, as the step (1-6), a progeny individual in which the DNAmarkers M1 and M5 are a homo-chromosome region of an allele derived fromthe original cultivar, and the DNA markers M2, M3, and M4 are ahomo-chromosome region of an allele derived from the foreign cultivar isselected, from the progeny individual obtained in the step (1-5); or aprogeny individual obtained by self-mating the progeny individualobtained in the step (1-5).

FIG. 2D to FIG. 2F are views showing a chromosome region of a progenyindividual preferable in the step (1-6) among progeny individualsobtained in the step (1-5). In the figures, a non-filled bold line showsan allele derived from the original cultivar, and a filled bold lineshows an allele derived from the foreign cultivar. First, from theprogeny individual obtained in the step (1-5), a progeny individual (1d)in which the DNA markers M1 and M5 are a homo-chromosome region of anallele derived from the original cultivar, and the DNA markers M2, M3and M4 are a hetero-chromosome region; a progeny individual (1e) inwhich the DNA markers M1 and M5 are a homo-chromosome region of anallele derived from the original cultivar, and the DNA markers M2, M3and M4 are a homo-chromosome region of an allele derived from theforeign cultivar; a progeny individual (1f) in which the DNA marker M1is a homo-chromosome region of an allele derived from the originalcultivar, the DNA markers M2, M3 and M4 are a homo-chromosome region ofan allele derived from the foreign cultivar, and the DNA marker M5 is ahetero-chromosome region; are selected, respectively. Herein, theprogeny individual (1e) is an objective new cultivar created by thefirst method of creating a new cultivar of the present invention, inwhich an end on an upstream side of a foreign cultivar-derivedchromosome fragment L is between the DNA markers M1 and M2, and an endon a downstream side thereof is between the DNA markers M4 and M5. Theprogeny individual (1d) and the progeny individual (1f) are a furtherself-mated, respectively, and the progeny individual (1e) can beselected from the resulting progeny individual.

Alternatively, for determining both ends of the target region, after anend on an upstream side of the foreign cultivar-derived chromosomefragment to be introduced is determined, an end of a downstream side maybe determined like the first method of creating a new cultivar of thepresent invention, or after an end of a downstream side is determined,an end on an upstream side may be determined like a second method ofcreating a new cultivar of the present invention described later.

In a second method of creating a new cultivar of the present invention,the following steps (2-1) to (2-6) are performed for every one targetregion on one or plural target regions in a chromosome of an originalcultivar, using a chromosome fragment-substituted line in which only apart of a chromosome of the original cultivar is substituted with achromosome fragment derived from a foreign cultivar.

(2-1) A step of defining a DNA marker M2 at an end on an upstream sideof a target region, or upstream thereof; a step of defining a DNA markerM1 upstream of the DNA marker M2; a step of defining a DNA marker M4 atan end on a downstream side of a target region, or a downstream thereof;a step of defining a DNA marker M5 downstream of the DNA marker M4; astep of defining a DNA marker M3 in the target region.(2-2) A step of mating a chromosome fragment-substituted line and anoriginal cultivar to obtain a progeny individual in which the DNA markerM3 is a hetero-chromosome region of an allele derived from an originalcultivar and an allele derived from a foreign cultivar.(2-3) A step of self-mating the progeny individual obtained in the step(2-2) to obtain a progeny individual.(2-4) A step of selecting a progeny individual in which the DNA markerM5 is a homo-chromosome region of an allele derived from the originalcultivar, and the DNA markers M3 and M4 are a hetero-chromosome regionof an allele derived from the original cultivar and an allele derivedfrom the foreign cultivar, from the progeny individual obtained in thestep (2-3); or a progeny individual obtained by backcrossing the progenyindividual obtained in the step (2-3).(2-5) A step of self-mating the progeny individual selected in the step(2-4) to obtain a progeny individual.(2-6) A step of selecting a progeny individual in which the DNA markersM1 and M5 are a homo-chromosome region of an allele derived from theoriginal cultivar, and the DNA markers M2, M3 and M4 are ahomo-chromosome region of an allele derived from the foreign cultivar,from the progeny individual obtained in the step (2-5); or a progenyindividual obtained by self-mating the progeny individual obtained inthe step (2-5).

The steps (2-1) to (2-3) are the same as the steps (1-1) to (1-3) of thefirst method of creating a new cultivar of the present invention,respectively.

FIG. 3A to FIG. 3C are views showing a chromosome region of a progenyindividual preferable in the step (2-4) among progeny individualsobtained in the step (2-3). In the figures, a non-filled bold line showsan allele derived from an original cultivar, and a filled bold lineshows an allele derived from a foreign cultivar, respectively. First,from the progeny individual obtained in the step (2-3), a progenyindividual (2a) in which the DNA marker M5 is a homo-chromosome regionof an allele derived from the original cultivar, and the DNA markers M4and M3 are a hetero-chromosome region; a progeny individual (2b) inwhich the DNA marker M5 is a hetero-chromosome region, and the DNAmarkers M4 and M3 are a homo-chromosome region of an allele derived fromthe foreign cultivar; a progeny individual (2c) in which the DNA markerM5 is a homo-chromosome region of an allele derived from the originalcultivar, and the DNA markers M4 and M3 are a homo-chromosome region ofan allele derived from the foreign cultivar; are selected, respectively.Herein, the progeny individual (2a) is a progeny individual finallyselected in the step (2-4). The progeny individual (2b) and the progenyindividual (2c) are further backcrossed with an individual of eachoriginal cultivar, and the progeny individual (2a) can be selected fromthe resulting progeny individual.

FIG. 3D to FIG. 3F are views showing a chromosome region of a progenyindividual preferable in the step (2-6) among progeny individualsobtained in the step (2-5). In the figures, a non-filled bold line showsan allele derived from the original cultivar, and a filled bold lineshows an allele derived from the foreign cultivar. First, from theprogeny individual obtained in the step (2-5), a progeny individual (2d)in which the DNA markers M1 and M5 are a homo-chromosome region of anallele derived from the original cultivar, and the DNA markers M2, M3and M4 are a hetero-chromosome region; a progeny individual (2e) inwhich the DNA markers M1 and M5 are a homo-chromosome region of anallele derived from the original cultivar, and the DNA markers M2, M3and M4 are a homo-chromosome region of an allele derived from theforeign cultivar; a progeny individual (2f) in which the DNA marker M5is a homo-chromosome region of an allele derived from the originalcultivar, the DNA markers M2, M3 and M4 are a homo-chromosome region ofan allele derived from the foreign cultivar, and the DNA markers M1 is ahetero-chromosome region; are selected, respectively. Herein, theprogeny individual (2e) is an objective new cultivar created by thesecond method of creating a new cultivar of the present invention, inwhich an end on an upstream side of a foreign cultivar-derivedchromosome fragment L is between the DNA markers M1 and M2, and an endon a downstream side thereof is between the DNA markers M4 and M5. Theprogeny individual (2d) and the progeny individual (2f) are furtherself-mated, respectively, and then the progeny individual (2e) can beselected from the resulting progeny individual.

Alternatively, for determining both ends of the target region, after aone side end of the foreign cultivar-derived chromosome fragment to beintroduced is determined, other end may be determined like the first orsecond method of creating an new cultivar of the present invention, orends on both sides may be determined first like a third method ofcreating a new cultivar of the present invention described later.

In a third method of creating a new cultivar of the present invention,the following steps (3-1) to (3-6) are performed for every one targetregion on one or plural target regions in a chromosome of an originalcultivar, using a chromosome fragment-substituted line in which only apart of a chromosome of the original cultivar is substituted with aforeign cultivar-derived chromosome fragment.

(3-1) A step of defining a DNA marker M2 at an end on an upstream sideof a target region, or upstream thereof; a step of defining a DNA markerM1 upstream of the DNA marker M2; a step of defining a DNA marker M4 atant end on a downstream side of the target region, or the downstreamthereof; a step of defining a DNA marker M5 downstream of the DNA makerM4; a step of defining a DNA marker M3 in the target region.(3-2) A step of mating a chromosome fragment-substituted line and anoriginal cultivar to obtain a progeny individual in which the DNA markerM3 is a hetero-chromosome region of an allele derived from the originalcultivar and an allele derived from a foreign cultivar.(3-3) A step of self-mating the progeny individual obtained in the step(3-2) to obtain a progeny individual.(3-4) A step of selecting a progeny individual in which any one of theDNA markers M1 and M5 is a homo-chromosome region of an allele derivedfrom the original cultivar, and the other is a hetero-chromosome regionof an allele derived from the original cultivar and an allele derivedfrom the foreign cultivar, from the progeny individual obtained in thestep (3-3); or a progeny individual obtained by backcrossing the progenyindividual obtained in the step (3-3).(3-5) A step of self-mating the progeny individual selected in the step(3-4) to obtain a progeny individual.(3-6) A step of selecting a progeny individual in which the DNA markersM1 and M5 are a homo-chromosome region of an allele derived from theoriginal cultivar, and the DNA markers M2, M3 and M4 are ahomo-chromosome region of an allele derived from the foreign cultivar,from the progeny individual obtained in the step (3-5); or a progenyindividual obtained by self-mating the progeny individual obtained inthe step (3-5).

The steps (3-1) to (3-3) are the same as the steps (1-1) to (1-3) of thefirst method of creating a new cultivar of the present invention.

As the step (3-4), a progeny individual in which any one of the DNAmarkers M1 and M5 is a homo-chromosome region of an allele derived fromthe original cultivar, and the other is a hetero-chromosome region of anallele derived from the original cultivar and an allele derived from aforeign cultivar, from the progeny individual obtained in the step(3-3); or a progeny individual obtained by backcrossing the progenyindividual obtained in the step (3-3). That is, from among progenyindividuals obtained in the step (3-3), an objective progeny individualmay be selected, or after a progeny individual in which in at least anyone of alleles, a recombination point about the originalcultivar-derived allele region and the foreign cultivar-derived alleleregion is present between the DNA markers M1 and M5 is selected fromprogeny individuals obtained in the step (3-3), an objective progenyindividual may be selected from among progeny individuals obtained bybackcrossing this progeny individual.

FIG. 4A to FIG. 4F are views showing a chromosome region of a progenyindividual preferable in the step (3-4) among progeny individualsobtained in the step (3-3). In the figures, a non-filled bold line showsan allele derived from the original cultivar, and a filled bold lineshows an allele derived from the foreign cultivar, respectively. First,from the progeny individual obtained in the step (3-3), a progenyindividual (3a) in which the DNA marker M1 is a homo-chromosome regionof an allele derived from the original cultivar, and the DNA marker M5is a hetero-chromosome region; a progeny individual (3b) in which theDNA marker M1 is a hetero-chromosome region, and the DNA marker M5 is ahomo-chromosome region of an allele derived from the foreign cultivar; aprogeny individual (3c) in which the DNA marker M1 is a homo-chromosomeregion of an allele derived from the original cultivar, and the DNAmarker M5 is a homo-chromosome region of an allele derived from theforeign cultivar; a progeny individual (3d) in which the DNA marker M1is a hetero-chromosome region, and the DNA marker M5 is ahomo-chromosome region of an allele derived from the original cultivar;a progeny individual (3e) in which the DNA marker M1 is ahomo-chromosome region of an allele derived from the foreign cultivar,and the DNA marker M5 is a hetero-chromosome region; a progenyindividual (3f) in which the DNA marker M1 is a homo-chromosome regionof an allele derived from the foreign cultivar, and the DNA marker M5 isa homo-chromosome region of an allele derived from the originalcultivar; are selected, respectively. Herein, the progeny individual(3a) or (3d) is a progeny individual finally selected in the step (3-4).The progeny individual (3b), (3c), (3e) or (3f) is further backcrossedwith an individual of each original cultivar, and the progeny individual(3a) or (3d) can be selected from the resulting progeny individual.

Then, as the step (3-5), by self-mating the progeny individual (3a) or(3d) selected in the step (3-4) like this, a progeny individual isobtained. Thereafter, as the step (3-6), a progeny individual in whichthe DNA markers M1 and M5 are a homo-chromosome region of an allelederived from the original cultivar, and the DNA markers M2, M3 and M4are a homo-chromosome region of an allele derived from the foreigncultivar is selected, from the progeny individual obtained in the step(3-5); or a progeny individual obtained by self-mating the progenyindividual obtained in the step (3-5).

FIG. 5A to FIG. 5C are views showing a chromosome region of a progenyindividual preferable in the step (3-6), among the case where theprogeny individual obtained in the step (3-4) is (3a). In the figures, anon-filled bold line shows an allele derived from the original cultivar,and a filled bold line shows an allele derived from the foreigncultivar. First, from progeny individuals obtained in the step (3-5), aprogeny individual (3g) in which the DNA markers M1 and M5 are ahomo-chromosome region of an allele derived from the original cultivar,and the DNA markers M2, M3 and M4 are a hetero-chromosome region; aprogeny individual (3h) in which the DNA markers M1 and M5 are ahomo-chromosome region of an allele derived from the original cultivar,and the DNA markers M2, M3 and M4 are a homo-chromosome region of anallele derived from the foreign cultivar; a progeny individual (3i) inwhich the DNA marker M1 is a homo-chromosome region of an allele derivedfrom the original cultivar, the DNA markers M2, M3 and M4 are ahomo-chromosome region of an allele derived from the foreign cultivar,and the DNA marker M5 is a hetero-chromosome region; are selected,respectively. Herein, the progeny individual (3h) is an objective newcultivar created by the third method of creating a new cultivar of thepresent invention, an end on an upstream side of a foreigncultivar-derived chromosome fragment L is between the DNA markers M1 andM2, and an end on a downstream side is between the DNA markers M4 andM5. The progeny individual (3g) and the progeny individual (3i) arefurther self-mated, respectively, and the progeny individual (3h) can beselected from the resulting progeny individual.

FIG. 5D to FIG. 5F are views showing a chromosome region of a chromosomeindividual preferable in the step (3-6), among the case where theprogeny individual obtained in the step (3-4) is (3d). In the figures, anon-filled bold line shows an allele derived from the original cultivar,and a filled bold line shows an allele derived from the foreigncultivar. First, from progeny individuals obtained in the step (3-5), aprogeny individual (3j) in which the DNA markers M1 and M5 are ahomo-chromosome region of an allele derived from the original cultivar,and the DNA markers M2, M3 and M4 are a hetero-chromosome region; aprogeny individual (3k) in which the DNA markers M1 and M5 are ahomo-chromosome region of an allele derived from the original cultivar,and the DNA markers M2, M3 and M4 are a homo-chromosome region of anallele derived from the foreign cultivar; a progeny individual (31) inwhich the DNA marker M5 is a homo-chromosome region of an allele derivedfrom the original cultivar, the DNA markers M2, M3 and M4 are ahomo-chromosome region of an allele derived from the foreign cultivar,and the DNA marker M1 is a hetero-chromosome; are selected,respectively. Herein, the progeny individual (3k) is an objective newcultivar created by the third method of creating a new cultivar of thepresent invention, an end on an upstream side of a foreigncultivar-derived chromosome fragment L is between the DNA markers M1 andM2, and an end on a downstream side is between the DNA markers M4 andM5. The progeny individual (3j) and (31) are further self-mated,respectively, and the progeny individual (3k) can be selected from theresulting progeny individual.

In the third method of creating a new cultivar of the present invention,even when all progeny individuals (3a) to (3f) shown in FIG. 4A to FIG.4F are selected in the step (3-4), by performing the following steps(3-7-1) and (3-7-2) before the step (3-5), an objective progenyindividual in which the DNA markers M1 and M5 are a homo-chromosomeindividual of an allele derived from the original cultivar, and the DNAmarkers M2, M3 and M4 are a homo-chromosome region of an allele derivedfrom the foreign cultivar can be obtained.

(3-7-1) A step of obtaining a progeny individual by self-mating theprogeny individual selected in the step (3-4).(3-7-2) A step of selecting (ii-1) a progeny individual in which the DNAmarker M1 is a homo-chromosome region of an allele derived from theoriginal cultivar, and the DNA markers M2 and M3 are a hetero-chromosomeregion of an allele derived from the original cultivar and an allelederived from the foreign cultivar; or (ii-2) a progeny individual inwhich the DNA marker M5 is a homo-chromosome region of an allele derivedfrom the original cultivar, and the DNA markers M3 and M4 are ahetero-chromosome region of an allele derived from the original cultivarand an allele derived from the foreign cultivar, from the progenyindividual obtained in the step (3-7-1); a progeny individual obtainedby backcrossing the progeny individual obtained in the step (3-7-1); ora progeny individual obtained by further backcrossing a progenyindividual obtained by self-mating the progeny individual obtained inthe step (3-7-1).

Among individuals selected in the step (3-7-2), (ii-1) the progenyindividual in which the DNA marker M1 is a homo-chromosome region of anallele derived from the original cultivar, and the DNA markers M2 and M3are a hetero-chromosome region of an allele derived from the originalcultivar and an allele derived from the foreign cultivar corresponds tothe progeny individual (1a) finally selected in the step (1-4) of thefirst method of creating a new cultivar of the present invention. On theother hand, from individuals selected in the step (3-7-2), (ii-2) theprogeny individual in which the DNA marker M5 is a homo-chromosomeregion of an allele derived from the original cultivar, and the DNAmarkers M3 and M4 are a hetero-chromosome region of an allele derivedfrom the original cultivar and an allele derived from the foreigncultivar corresponds to the progeny individual (2a) finally selected inthe step (2-4) of the second method of creating a new cultivar of thepresent invention. Therefore, the individual selected on the step(3-7-2) is a progeny individual in which an end on an upstream side of aforeign cultivar-derived chromosome fragment L is between the DNAmarkers M1 and M2, and an end on a downstream side thereof is betweenthe DNA markers M4 and M5, by performing the step (3-5′) and (3-6′),like the steps (1-5) and (1-6) of the first method of creating a newcultivar of the present invention, or the steps (2-5) and (2-6) of thesecond method of creating a new cultivar of the present invention. Thatis, by selection in the step (3-7-2), an objective new cultivar createdby the third method of creating a new cultivar of the present inventioncan be obtained.

FIG. 6A to FIG. 9G are views showing a chromosome region of a relativelypreferable progeny individual among progeny individuals obtained in thestep (3-7-1). In the figures, a non-filled bold line shows an allelederived from the original cultivar, and a filled bold line shows anallele derived from the foreign cultivar. A progeny individual (3a-a) inwhich the DNA marker M1 is a homo-chromosome region of an allele derivedfrom the original cultivar, and the DNA markers M2 to M5 is ahetero-chromosome region; and a progeny individual (3a-b) in which theDNA markers M1 and M2 are a homo-chromosome region of an allele derivedfrom the original cultivar, and the DNA markers M3 to M5 are ahetero-chromosome region; are a progeny individual obtained byself-mating of the progeny individual (3a) (see FIG. 6A and FIG. 6B).

A progeny individual (3b-a) in which the DNA markers M1 and M2 are ahetero-chromosome region, and the DNA markers M3 to M5 are ahomo-chromosome region of an allele derived from the foreign cultivar;and a progeny individual (3b-b) in which the DNA marker M1 is ahetero-chromosome region, and the DNA markers M2 to M5 are ahomo-chromosome region of an allele derived from the foreign cultivar;are a progeny individual obtained by self-mating of the progenyindividual (3b) (see FIG. 6C and FIG. 6D).

A progeny individual (3c-a) in which the DNA marker M1 is ahomo-chromosome region of an allele derived from the original cultivar,the DNA markers M2 to M4 are a hetero-chromosome region, and the DNAmarker M5 is a homo-chromosome region of an allele derived from theforeign cultivar; a progeny individual (3c-b) in which the DNA marker M1is a homo-chromosome region of an allele derived from the originalcultivar, and the DNA markers M2 to M5 are a homo-chromosome region ofan allele derived from the foreign cultivar; a progeny individual (3c-c)in which the DNA marker M1 is a homo-chromosome region of an allelederived from the original cultivar, the DNA marker M2 is ahetero-chromosome region, and the DNA markers M4 and M5 are ahomo-chromosome region of an allele derived from the foreign cultivar; aprogeny individual (3c-d) in which the DNA marker M1 is ahomo-chromosome region of an allele derived from the original cultivar,the DNA markers M2 and M3 are a hetero-chromosome region, and the DMAmakers M4 and M5 are a homo-chromosome region of an allele derived fromthe foreign cultivar; a progeny individual (3c-e) in which the DNAmarker M1 is a homo-chromosome region of an allele derived from theoriginal cultivar, the DNA markers M2 to M4 are a hetero-chromosomeregion, and the DNA marker M5 is a homo-chromosome region of an allelederived from the foreign cultivar; a progeny individual (3c-f) in whichthe DNA markers M1 and M2 are a homo-chromosome region of an allelederived from the original cultivar, the DNA marker M3 is ahetero-chromosome region, and the DNA markers M4 and M5 are ahomo-chromosome region of an allele derived from the foreign cultivar;and a progeny individual (3c-g) in which the DNA markers M1 and M2 are ahomo-chromosome region of an allele derived from the original cultivar,the DNA markers M3 and M4 are a hetero-chromosome region, and the DNAmarker M5 is a homo-chromosome region of an allele derived from theforeign cultivar; are a progeny individual obtained by self-mating ofthe progeny individual (3c) (see FIG. 7A to FIG. 7G).

In addition, a progeny individual (3d-a) in which the DNA markers M1 toM4 are a hetero-chromosome region, and the DNA marker M5 is ahomo-chromosome region of an allele derived from the original cultivar;and a progeny individual (3d-b) in which the DNA markers M1 to M3 are ahetero-chromosome region, and the DNA markers M4 and M5 are ahomo-chromosome region of an allele derived from the original cultivar;are a progeny individual obtained by self-mating of the progenyindividual (3d) (see FIG. 8A and FIG. 8B).

A progeny individual (3e-a) in which the DNA markers M1 to M4 are ahomo-chromosome region of an allele derived from the foreign cultivar,and the DNA marker M5 is a hetero-chromosome region; and a progenyindividual (3e-b) in which the DNA markers M1 to M3 are ahomo-chromosome region of an allele derived from the foreign cultivar,and the DNA markers M4 and M5 are a hetero-chromosome region; are aprogeny individual obtained by self-mating of the progeny individual(3e) (see FIG. 8C and FIG. 8D).

A progeny individual (3f-a) in which the DNA marker M1 is ahomo-chromosome region of an allele derived from the original cultivar,the DNA markers M2 to M4 are a hetero-chromosome region, and the DNAmarker M5 is a homo-chromosome region of an allele derived from theforeign cultivar, a progeny individual (3f-b) in which the DNA markersM1 to M4 are a homo-chromosome region of an allele derived from theforeign cultivar, and the DNA marker M5 is a homo-chromosome region ofan allele derived from the original cultivar; a progeny individual(3f-c) in which the DNA markers M1 and M2 are a homo-chromosome regionof an allele derived from the foreign cultivar, the DNA marker M4 is ahetero-chromosome region, and the DNA marker M5 is a homo-chromosomeregion of an allele derived from the original cultivar; a progenyindividual (3f-d) in which the DNA markers M1 and M2 are ahomo-chromosome region of an allele derived from the foreign cultivar,the DNA markers M3 and M4 are a hetero-chromosome region, and the DNAmarker M5 is a homo-chromosome region of an allele derived from theoriginal cultivar; a progeny individual (3f-e) in which the DNA markerM1 is a homo-chromosome region of an allele derived from the foreigncultivar, the DNA markers M2 to M4 are a hetero-chromosome region, andthe DNA marker M5 is a homo-chromosome region of an allele derived fromthe original cultivar; a progeny individual (3f-f) in which the DNAmarkers M1 and M2 are a homo-chromosome region of an allele derived fromthe foreign cultivar, the DNA marker M3 is a hetero-chromosome region,and the DNA markers M4 and M5 are a homo-chromosome region of an allelederived from the original cultivar; and a progeny individual (3f-g) inwhich the DNA marker M1 is a homo-chromosome region of an allele derivedfrom the foreign cultivar, the DNA markers M2 and M3 are ahetero-chromosome region, and the DNA markers M4 and M5 are ahomo-chromosome region of an allele derived from the original cultivar;are a progeny individual obtained by self-mating of the progenyindividual (3f) (see FIG. 9A to FIG. 9G).

Among these progeny individuals obtained in the step (3-7-1), theprogeny individual (3a-a) corresponds to the progeny individual (1a)shown in FIG. 2A, and the progeny individual (3d-a) corresponds to theprogeny individual (2a) shown in FIG. 3A. Therefore, these progenyindividuals can be selected to proceed to the next step (3-5′).

In addition, when the progeny individuals (3b-b), (3c-a), (3c-b),(3c-c), (3c-d), and (3c-e) are self-mated, respectively, an individualin which a chromosome region corresponds to that of the progenyindividual (1a) can be included in progeny individuals obtained by thisself-mating. Similarly, when the progeny individuals (3e-a), (3f-a),(3f-b), (3f-c), (3f-d), and (3f-e) are self-mated, respectively, anindividual in which a chromosome region corresponds to that of theprogeny individual (2a) is included in progeny individual obtained bythis self-mating. Then, these progeny individuals can be selected toproceed to a next step (3-5′).

On the other hand, when the progeny individual (3b-a) is self-mated,among progeny individuals obtained by this self-mating, an individual inwhich a chromosome region corresponds to that of the progeny individual(3b-b) can be contained. Similarly, when the progeny individual (3e-b)is self-mated, among progeny individuals obtained by this self-mating,an individual in which a chromosome region corresponds to that of theprogeny individual (3e-a) can be contained. Then, these progenyindividuals corresponding to the progeny individual (3b-b) or (3e-a) areselected from the self-mated groups, and further self-mated. And then,among progeny individuals obtained by this self-mating, an individual inwhich a chromosome region corresponds to that of the progeny individual(1a) or the progeny individual (2a) can be selected to proceed to thenext step (3-5′).

In progeny individuals selected in the step (3-4), a progeny individualin which a position of a recombination point about a region of an allelederived from the original species and a region of an allele derived fromthe foreign cultivar is unknown, and a target region is not substitutedwith a chromosome fragment derived from the foreign cultivar; and aprogeny individual in which a target region is merely partiallysubstituted with a chromosome fragment derived from the foreigncultivar; are also contained. Then, from progeny individuals selected inthe step (3-4), a progeny individual in which the target region issubstituted with a chromosome fragment derived from the foreign cultivaris selected, and this selected progeny individual may be used in thestep (3-5).

Herein, selection of a progeny individual in which the target region issubstituted with a chromosome fragment derived from the foreign cultivarmay be a selection using a DNA marker, or may be a selection by thecharacter detection. In the case of selection by using the DNA marker, aprogeny individual in which a DNA marker M3 is a hetero-chromosomeregion of an allele derive from the original cultivar and an allelederived from the foreign cultivar is selected. In the case of selectionby the character detection, a progeny individual having a targetcharacter introduced by substitution with a foreign cultivar-derivedchromosome fragment is selected. When the number of the progenyindividuals obtained in the step (3-3) is small, selection by thecharacter detection may be performed.

It is preferable to confirm whether progeny individuals selected in thestep (1-6), (2-6) or (3-6), that is, new cultivars created by the firstto third methods of creating a new cultivar of the present inventionhave an objective character or not. For example, autogamous seeds arecollected from individuals of a new cultivar, and the seeds areindividually cultivated as a population. By appropriately observing oranalyzing the cultivated populations, possession of an objectivecharacter and no segregation of the population as a whole are confirmed.

In addition, in the first to third methods of creating a new cultivar ofthe present invention, the number of the target regions may be one orplural. In the case of plural target regions, a progeny individual inwhich all target regions are substituted with a homo-chromosome fragmentderived from the foreign cultivar can be obtained by repeatedlyperforming the above steps for every target region.

According to the first to third methods of creating a new cultivar ofthe present invention, a region of a chromosome fragment derived fromthe foreign cultivar to be introduced into a chromosome of the originalcultivar can be controlled, and other genes other than an objective generegion can be effectively suppressed from being introduced into achromosome of the original cultivar. Therefore, a new cultivar having atarget character can be made without changing a preferable characterpossessed by the original cultivar. For this reason, in cultivarscreated by the first to third methods of creating a new cultivar of thepresent invention, the effect of improving a character of the originalcultivar by a chromosome fragment introduced into the chromosome can bedetermined with very high reliance.

According to the first to third methods of creating a new cultivar ofthe present invention, via a specified step such as the steps (1-1) to(1-6), a cultivar in which a region encoding an objective gene is atarget region, and only such a short region that other genes other thanthis objective gene are not contained, is substituted with a chromosomefragment derived from the foreign cultivar can be created.

For example, in a rice genome, theoretically, unless the region ofchromosome is 12 Mbp or more as an average, crossing of the chromosomesoccur simultaneously at 2 points, and a recombinant in which this regionis recombined cannot be obtained theoretically, due to a chiasmainterference. Therefore, an existing probability of a progeny individualin which only a short specified region is substituted is very small. Forthis reason, when a DNA marker is merely set for a desired region andthe selection is performed by the previous MAS method using this DNAmarker as an index, it is necessary that a selected population is largescale. For this reason, the labor and the cost required for theselection become excessive. Further, since an amount of a seed which canbe harvested from one rice is limited, there is a high possibility thatthe desired progeny individual cannot be obtained even when screening isrepeated many times.

To the contrary, the first to third methods of creating a new cultivarof the present invention allow for creation of a progeny individual inwhich only a chromosome fragment region is substituted just as designedas keeping a selection population being a general size.

In addition, in the first to third methods of creating a new cultivar ofthe present invention, respective DNA markers M1 to M5 defined for thetarget region are genome information peculiar to cultivars created bythese methods. Therefore, the cultivars created by first to thirdmethods of creating a new cultivar of the present invention can bediscriminated using these DNA markers.

Specifically, a method of discriminating a plant cultivar of the presentinvention is a method of discriminating whether a plant individual is aspecified cultivar produced using the first to third methods of creatinga new cultivar of the present invention, wherein one or more DNA markersselected from the group consisting of DNA markers M1 to M5 are typed bygenome analysis of this plant individual and, when the typing result isconsistent with the result of the specified cultivar, it is determinedthat this plant individual is the specified cultivar.

Herein, five of DNA markers M1 to M5 are designed for every one targetregion, but all of these DNA markers M1 to M5 may be used, and some ofthese DNA markers may be used, for discriminating a cultivar. Forexample, only DNA markers M1 and M2 which are a recombination point onan upstream side of the target region may be used, only the DNA markersM4 and M5 which are a recombination point on a downstream side of atarget region may be used, or only DNA markers M2 and M4 containing thetarget region therebetween may be used. Alternatively, when the numberof target regions is plural, DNA markers of respective target regionsmay be appropriately used by combination. By appropriately combiningplural DNA markers, it becomes possible to discriminate cultivar morestrictly.

Individuals of cultivars created by the first to third methods ofcreating a new cultivar of the present invention (hereinafter, referredto as present first cultivar in some cases) can be mated to obtain aprogeny individual like an individual of the original cultivar used increation of this individual. Particularly, it is preferable to obtain aprogeny individual by mating two individuals selected from the groupconsisting of an individual of the present first cultivar and a progenyindividual of this first cultivar. In the present invention, it ispreferable that these two individuals are such that at least one targetregion is different from each other. In addition, it is preferable thata progeny individual obtained by mating is such that plural targetregions in a chromosome of the original cultivar are substituted with ahomo-chromosome fragment derived from the foreign cultivar.

Then, a fourth method of creating a new cultivar of the presentinvention will be explained. In the fourth method of creating a newcultivar of the present invention, among individuals of the presentfirst cultivars created by the first to third methods of creating a newcultivar of the present invention, two individuals in which at least onetarget region is different from each other are mated as a parentindividual. Thereby, a new cultivar having a genome in which regionssubstituted with a chromosome fragment derived from the foreign cultivarpossessed by each parent individual are accumulated, can be created.

That is, the fourth method of crating a new cultivar of the presentinvention has steps: (4-1) a step of using an individual of the presentfirst cultivar as a seed parent, and an individual of the present firstcultivar in which at least one target region is different from this seedparent as a pollen parent, and mating the seed parent and the pollenparent to obtain a progeny individual; (4-2) a step of self-mating theprogeny individual obtained in the step (4-1) to obtain a progenyindividual; (4-3) a step of selecting a progeny individual in which, ina chromosome of the original cultivar, all of target regions possessedby the seed parent and target regions possessed by the pollen parent aresubstituted with a homo-chromosome derived from the foreign cultivar,from the progeny individual obtained in the step (4-2).

In addition, the fourth method of creating a new cultivar of presentinvention may have further steps after the step (4-3): (4-4) a step ofselecting two individuals in which at least one target region isdifferent from each other, as a seed parent and a pollen parent, fromthe group consisting of an individual of the present first cultivar andan individual selected in the step (4-3), and mating them to obtain aprogeny individual; (4-5) a step of self-mating the progeny individualobtained by the step (4-4) to obtain a progeny individual; (4-6) a stepof selecting a progeny individual in which, in a chromosome of theoriginal cultivar, all of a target region possessed by the seed parentand a target region possessed by the pollen parent are substituted witha homo-chromosome fragment derived from the foreign cultivar, from theprogeny individuals obtained by the step (4-5); (4-7) a step ofrepeating the steps (4-4) to (4-6) once or more.

In addition, in each progeny individual obtained by the fourth method ofcreating a new cultivar of the present invention, whether each targetregion is a homozygote fragment derived from the foreign cultivar can bediscriminated using DNA markers M1 to M5 used in the method of creatingthe first cultivar of the present invention.

In the previous cultivar improving method by backcrossing and the MASmethod, a length of a chromosome fragment to be introduced in achromosome of the original cultivar cannot be controlled, as describedabove. For this reason, many genes having the unknown function inaddition to an objective gene are introduced together into a chromosomeof the original cultivar. As the number of introduced chromosomefragment is increased, the number of introduced genes having unknownfunction is increased. Therefore, when one try to improve a plurality ofcharacters by mating, a problem such as deterioration in character otherthan an objective character to be improved arises. In addition, likethis, since there is a high possibility that many unknown genes areintroduced, an objective character is not necessarily improved with anintentionally introduced chromosome fragment (chromosome fragmentcontaining target region). For this reason, even in the case of aprogeny individual having a chromosome fragment of a target region, manyindividuals having unimproved objective characters are obtained.Further, DNA markers used in selection are merely linked with achromosome fragment of the target region in the original cultivar. Forthis reason, the chromosomes are randomly arranged by plural timesmating of plant individuals and, as a result, DNA markers become not tolink with a chromosome fragment of the target region. Therefore, whenthis DNA marker is used, a progeny individual having a chromosomefragment of the target region cannot be selected in many cases.

For example, by the previous mating method, an individual P1 (A) inwhich a homo-chromosome fragment of a target region A derived from theforeign cultivar is introduced into a chromosome of the originalcultivar, and an individual P1 (B) in which a homo-chromosome fragmentof a target region B derived from the foreign cultivar is introducedinto a chromosome of the original cultivar are mated, and the resultingprogeny individual is self-mated. Thereby, an individual P2 (AB) inwhich both of target regions A and B derived from the foreign cultivarare a homozygote, in a chromosome of the original cultivar, is obtained.Thereupon, when the target regions A and B are independent from eachother, and are subject to a Mendel's law, the individual P2 (AB) can beselected from a progeny individual obtained by self-mating theoreticallyat a probability of 1/16. However, the selected individual P2 (AB) isnot necessarily improved in objective two characters and, althoughobjective characters are improved, other characters are deteriorated inmany cases. This problem is more serious as the number of introductiontarget regions becomes larger and, actually, it was very difficult toimprove three or more characters.

To the contrary, in individuals of cultivars created by the first tothird methods of creating a new cultivar of the present invention, andprogeny individuals of them, introduction of a chromosome region into aregion other than the target region can be suppressed as much aspossible. For this reason, there is a very high possibility that acharacter different from that of the original cultivar is the effect ofthe introduced chromosome fragment of the target region derive from theforeign cultivar. Therefore, like the fourth method of creating a newcultivar of the present invention, for example, when an individual P1(A) in which a homo-chromosome fragment of a target region A derivedfrom the foreign cultivar is introduced into a chromosome of theoriginal cultivar by the first to third methods of creating a newcultivar of the present invention; and an individual P1 (B) in which ahomo-chromosome fragment of a target region B derived from the foreigncultivar is similarly introduced; are mated, to thereby an individual P2(AB) in which both of the target regions A and B derived from theforeign cultivar are a homozygote is obtained. In this individual P2(AB), it can be sufficiently expected that both characters of theimproved character A possessed by the individual P1 (A) and the improvedcharacter B possessed by the individual P1 (B) are improved withoutchanging a preferable character possessed by other original cultivars.Like this, by using the fourth method of creating a new cultivar of thepresent invention, improved characters can be sequentially accumulatedby mating, and three or more characters can be improved simply and at ahigh procession.

In addition, DNA markers M1 to M5 used in selection are DNA markers in atarget region or in vicinity of the target region. For this reason, evenwhen mating is repeated plural times like the case using the fourthmethod of creating a new cultivar of the present invention, a progenyindividual having a chromosome fragment of the target region can besufficiently selected using these DNA markers M1 to M5.

For example, an individual P1 (A) which is an individual of the presentfirst cultivar and in which a homo-chromosome fragment of a targetregion A derived from the foreign cultivar is introduced into achromosome of the original cultivar is used as a seed parent, and anindividual P1 (B) which is an individual of the present first cultivarand in which a homo-chromosome fragment of a target region B derivedfrom the foreign cultivar is introduced into a chromosome of theoriginal cultivar is used as a pollen parent. And, P1 (A) and P1 (B) aremated, the resulting progeny individual is self-mated to obtain aprogeny individual. And then, from the progeny individual obtained bythis self-mating, an individual P2 (AB) in which, in a chromosome of theoriginal cultivar, all of the target regions A and B are substitutedwith a homo-chromosome fragment derived from the foreign cultivar isselected. Thereby, a new cultivar can be created. Herein, when thetarget regions A and B are independent of each other without linkage,and are subject to a Mendel's law, the individual P2 (AB) can beselected from a progeny individual obtained by self-mating at aprobability of 1/16.

In addition, the thus obtained progeny individual P2 (AB), and anindividual P1 (C) in which a homo-chromosome fragment of a target regionC derived from the foreign cultivar is introduced into a chromosome ofthe original cultivar are mated to obtain a progeny individual.Thereafter, this obtained progeny individual is self-mated and, from theprogeny individual obtained by this self-mating, an individual P3 (ABC)in which, in a chromosome of the original cultivar, all of targetregions A, B and C are substituted with a homo-chromosome fragmentderived from the foreign cultivar is selected. Thereby, a new cultivarcan be created. Herein, when the target regions A, B and C areindependent of each other without linkage, and are subject to a Mendel'slaw, the individual P3 (ABC) can be selected from a progeny individualobtained by self-mating at a probability of 1/64.

The individual P3 (ABC) in which all of the target regions A, B and Care substituted with a homo-chromosome fragment derived from the foreigncultivar can be also created, for example, by the following method.First, P1 (B) and P1 (C) are mated to obtain a progeny individual, andthe resulting progeny individual is self-mated. From a progenyindividual obtained by this self-mating, an individual P2 (BC) in which,in a chromosome of the original cultivar, target regions B and C aresubstituted with a homo-chromosome fragment derived from the foreigncultivar is selected. Herein, when the target regions B and C areindependent of each other without linkage, and are subject to a Mendel'slaw, the individual P2 (BC) can be selected from progeny individualsobtained by self-mating at a probability of 1/16. Thereafter, P2 (AB)and P2 (BC) are mated to obtain a progeny individual, and then thisprogeny individual is self-mated. From the resulting progeny individual,P3 (ABC) is selected, thereby, P3 (ABC) can be created. In both of P2(AB) and P2 (BC), the target region B is a homozygote derived from theforeign cultivar. Therefore, when the target regions A, B and C areindependent respectively without linkage, and are subject to a Mendel'slaw, the individual P3 (ABC) can be selected from the progeny individualobtained by self-mating at a probability of 1/16.

FIG. 10A and FIG. 10B are a schematic view showing a method of creatinga cultivar in which, in a chromosome of the original cultivar, threetarget regions (target regions A, B, C) are substituted with achromosome fragment derived from the foreign cultivar. In the figures, asquare indicates each individual, and an alphabet in the squareindicates that each target region is substituted with a homo-chromosomefragment derived from the foreign cultivar. In a pyramid in whichsquares are piled, one square is piled on an upper step of two squares.This means that two squares on a lower step are a parent individual, andone square on an upper step is a progeny individual obtained by mating.In addition, a numerical value on a left side of the pyramid in whichsquares are piled indicates a probability of obtaining of each progenyindividual when respective target regions are independent respectivelywithout linkage, and are subject to a Mendel's law.

FIG. 10A shows a method of creating P3 (ABC) by mating theaforementioned P2 (AB) and P1 (C). FIG. 10B shows a method of creatingP3 (ABC) by mating the aforementioned P2 (AB) and P2 (BC). Like this, bysequentially mating individuals in which at least one target region isdifferent from each other among cultivars created by using the first tothird methods of creating a new cultivar of the present invention andprogeny individuals thereof, plural kinds of target regions substitutedwith a chromosome fragment derived from the foreign cultivar are beingaccumulated in a progeny individual. For this reason, a cultivar inwhich, in a chromosome of the original cultivar, four or more targetregions are substituted with a chromosome fragment derived from theforeign cultivar can be also created.

Herein, the case of production of a cultivar P4 (ABCD) in which, in achromosome of the original cultivar, four target regions (target regionsA, B, C, D) are substituted with a chromosome fragment derived from theforeign cultivar will be described. First, for example, an individual P2(AB) in which target regions A and B are substituted with ahomo-chromosome fragment derived from the foreign cultivar, and anindividual P2 (CD) in which target regions C and D are substituted witha homo-chromosome fragment derived from the foreign cultivar are matedto obtain a progeny individual. Then, by selecting P4 (ABCD) fromprogeny individuals obtained by self-mating this progeny individual, P4(ABCD) can be created. Thereupon, when target regions A, B, C and D areindependent respectively without linkage, and are subject to a Mendel'slaw, an individual P4 (ABCD) can be selected from a progeny individualobtained by the self-mating, at a probability of 1/256. Similarly, inthe case of creation of a cultivar P5 (ABODE) in which five targetregions (target regions A, B, C, D, E) are substituted with a chromosomefragment derived from the foreign cultivar, first, for example, anindividual P3 (ABC) in which target regions A, B, C are substituted witha homo-chromosome fragment derived from the foreign cultivar, and anindividual P2 (BD) in which target regions B and E are substituted witha homo-chromosome fragment derived from the foreign cultivar are matedto obtain a progeny individual and this progeny individual isself-mated. Then, by selecting P5 (ABODE) from progeny individualsobtained by this self-mating, P5 (ABODE) can be created. Thereupon, whentarget regions A, B, C, D and E are independent respectively withoutlinkage, and are subject a Mendel's law, the individual P5 (ABODE) canbe selected from a progeny individual obtained by self-mating, at aprobability of 1/1024.

However, usually, when an objective progeny individual is obtained, asize of a selected population (group of progeny individuals obtained byself-mating) is set to be around a few to 10-fold an existingprobability of an objective progeny individual. When a size of theselected population is insufficient, there is a high possibility that anobjective progeny individual is not obtained. On the other hand,usually, the number of seeds which can be collected from one individualis limited. For example, in rice, only around 1,000 seeds can be ensuredfrom one individual. In addition, a rice plant is weak, and sometimesonly a few tens of seeds are obtained from one plant. Further, as a sizeof the selected population grows larger, the necessary time, labor andcost become excessive. For this reason, it is thought that a productionmethod in which an existing probability of an objective progenyindividual is 1/1024 or more is actually very difficult to beimplemented.

In the fourth method of creating a new cultivar of the presentinvention, by sequentially mating the selected progeny individual, atarget region substituted with a chromosome fragment derived from theforeign cultivar can be accumulated in that progeny individual.Therefore, a new cultivar can be created so that an existing probabilityof an objective progeny individual in a once selected population becomes1/256 to 1/16.

For example, regarding the case where target regions A, B, C and D areindependent respectively without linkage, and are subject to a Mendel'slaw, a method of creating P4 (ABCD) will be described (see FIG. 11A toFIG. 11C). First, P2 (AB) and P2 (CD) are crated according to the samemanner as that of the aforementioned method of creating P2 (AB).Thereafter, P2 (AB) and P2 (CD) are mated to obtain a progenyindividual, and this progeny individual is self-mated. Then, byselecting P4 (ABCD) from progeny individuals obtained by thisself-mating, P4 (ABCD) can be created (see FIG. 11A). In this case,theoretically, P4 (ABCD) can be selected from a selected population at aprobability of 1/256.

Alternatively, P2 (AB), and P3 (BCD) created according to the samemanner as that of the aforementioned method of creating P3 (ABC) aremated to obtain a progeny individual, and this progeny individual isself-mated. From the progeny individual obtained by this self-mating, P4(ABCD) may be selected (see FIG. 11B). Both of P2 (AB) and P3 (BCD) aresuch that the target region B is a homozygote derived from the foreigncultivar. For this reason, theoretically, P4 (ABCD) can be selected froma selected population at a probability of 1/64.

Further, after P3 (ABC) and P3 (BCD) created according to the samemanner as that of P3 (BCD) are mated to obtain a progeny individual, P4(ABCD) may be selected from progeny individuals obtained by self-matingthis progeny individual (see FIG. 11C). Both of P3 (ABC) and P3 (BCD)are such that target regions B and C are a homozygote derived from theforeign cultivar. For this reason, theoretically, P4 (ABCD) can beselected from a selected population at a probability of 1/16.

That is, according to the method of creating a new cultivar of thepresent invention, even when the number of target regions is 4, anobjective new cultivar can be selected at a higher probability thanprevious.

In addition, for example, regarding the case where target regions, A, B,C, D and E are independent respectively without linkage, and are subjectto a Mendel's law, a method of creating P5 (ABCDE) will be described(see FIG. 12A to FIG. 12D). First, P2 (AB) and P3 (CDE) are mated toobtain a progeny individual and this progeny individual is self-mated.Thereafter, by selecting P5 (ABODE) from progeny individuals obtained bythis self-mating, P5 (ABCDE) can be created (see FIG. 12A). In thiscase, theoretically, P5 (ABCDE) can be selected from a selectedpopulation at a probability of 1/1024.

To the contrary, in the following case, a probability of selection of 25(ABCDE) is improved.

P3 (ABC) and P3 (CDE) are created according to the same manner as theaforementioned method of creating P3 (ABC), respectively. Thereafter,these P3 (ABC) and P3 (CDE) are mated to obtain a progeny individual andthis progeny individual is self-mated. Further, by selecting P5 (ABODE)from progeny individuals obtained by this self-mating, P5 (ABCDE) can becreated (see FIG. 12B). Both of P3 (ABC) and P3 (CDE) are such that atarget region C is a homozygote derived from the foreign cultivar. Forthis reason, in this case, theoretically, P5 (ABCDE) can be selectedfrom a selected population at a probability of 1/256.

Alternatively, P3 (ABC) is created according to the same manner as theaforementioned method of creating P3 (ABC), and P4 (BCDE) is createdaccording to the same manner as the aforementioned method of creating P4(ABCD). Thereafter, these P3 (ABC) and P4 (BODE) are mated to obtain aprogeny individual and this progeny individual is self-mated. Fromprogeny individuals obtained by this self-mating, P5 (ABCDE) may beselected (see FIG. 12C). Both of P3 (ABC) and P4 (BCDE) are such thattarget regions B and C are a homozygote derived from the foreigncultivar. For this reason, theoretically, P5 (ABCDE) can be selectedfrom a selected population at a probability of 1/64.

Besides, according to the same manner as the aforementioned method ofcreating P4 (ABCD), P4 (ABCD) and P4 (BCDE) are created, respectively.Thereafter, these P4 (ABCD) and P4 (BCDE) are mated to obtain a progenyindividual and this progeny individual is self-mated. Further, fromprogeny individuals obtained by this self-mating, P5 (ABCDE) may beselected (see FIG. 12D). Both of P4 (ABCD) and P4 (BODE) are such thattarget regions B, C and D are a homozygote derived from the foreigncultivar. For this reason, in this case, theoretically, P5 (ABCDE) canbe selected from a selected population at a probability of 1/16.

That is, according to the method of creating a new cultivar of thepresent invention, even when the number of target regions is 5, anobjective new cultivar can be selected at a higher probability thanprevious.

In addition, for example, regarding the case where target regions A, B,C, D, E and F are independent respectively without linkage, and aresubject to a Mendel's law, a method of creating P6 (ABCDEF) will bedescribed (see FIG. 13A to FIG. 13E). For example, after P3 (ABC) and P3(DEF) are mated to obtain a progeny individual, this progeny individualis self-mated. By selecting P6 (ABCDEF) from progeny individualsobtained by this self-mating, P6 (ABCDEF) can be created (see FIG. 13A).In this case, theoretically, a probability that P6 (ABCDEF) can beselected from a selected population is 1/4096.

Alternatively, after P3 (ABC) and P4 (CDEF) are mated to obtain aprogeny individual, this progeny individual is self-mated to obtainprogeny individuals. By selecting P6 (ABCDEF) from the progenyindividuals obtained by the self-mating, P6 (ACBDEF) can be also created(see FIG. 13B). In this case, theoretically, a probability that P6(ABCDEF) can be selected from a selected population is 1/1024.

To the contrary, in the following case, a probability of selection of P6(ABCDEF) is improved.

First, P4 (ABCD) and P4 (CDEF) are created according to the same manneras the aforementioned method of creating P4 (ABCD), respectively. Then,these P4 (ABCD) and P4 (CDEF) are mated to obtain a progeny individualand this progeny individual is self-mated. Further, by selecting P6(ABCDEF) from progeny individuals obtained by this self-mating, P6(ABCDEF) can be created (see FIG. 13C). Thereupon, theoretically, P6(ABCDEF) can be selected from a selected population at a probability of1/256. This is because both of P4 (ABCD) and P4 (CDEF) are such thattarget regions C and D are a homozygote derived from the foreigncultivar.

Alternatively, P4 (ABCD) is created according to the same manner as theaforementioned method of creating P4 (ABCD), and P5 (BCDEF) is createdaccording to the same manner as the aforementioned method of creating P5(ABCDE). Thereafter, after these P4 (ABCD) and P5 (BCDEF) are mated toobtain a progeny individual, this progeny individual is self-mated. Byselecting P6 (ABCDEF) from progeny individuals obtained by thisself-mating, P6 (ABCDEF) can be also created (see FIG. 13D). Both of P4(ABCD) and P5 (BCDEF) are such that target regions B, C and D are ahomozygote derived from the foreign cultivar. For this reason, in thiscase, theoretically, P5 (ABCDE) can be selected from a selectedpopulation at a probability of 1/64.

Alternatively, P5 (ABCDE) and P5 (BCDEF) are created according to thesame manner as the aforementioned method of creating P5 (ABCDE),respectively. Thereafter, after P5 (ABCDE) and P5 (BCDEF) are mated toobtain a progeny individual, this progeny individual is self-mated. Byselecting P6 (ABCDEF) from progeny individuals obtained by thisself-mating, P6 (ABCDEF) can be also created (see FIG. 13E). Both of P5(ABCDE) and P5 (BCDEF) are such that target regions B, C, D and E are ahomozygote derived from the foreign cultivar. For this reason, in thiscase, theoretically, P6 (ABCDEF) can be selected from a selectedpopulation at a probability of 1/16.

That is, according to the method of creating a new cultivar of thepresent invention, even when the number of target regions is 6, anobjective new cultivar can be selected at a higher probability thanprevious.

As described above, in the case of a plant in which the number ofprogeny individuals obtained by one time mating is relatively small,such as rice, it is preferable to create a new cultivar so that anexisting probability of an objective progeny individual in a one timeselected population becomes 1/64 to 1/16. That is, by sequentiallymating individuals of a combination in which a sum of different targetregions in a chromosome is 3 or less, it can be stably produced acultivar in which plural target regions in a chromosome of the originalcultivar are substituted with a chromosome fragment derived from theforeign cultivar.

FIG. 14A to FIG. 14C are schematic views showing a method of creating acultivar P6 (ABCDEF) as an existing probability of an objective progenyindividual in a one time selected population is 1/64 to 1/16. FIG. 14Ais a view showing a method when the existing probability in all selectedpopulations is 1/16. FIG. 14B and FIG. 14C are a view showing a methodwhen the existing probability in all selected populations is 1/16 or1/64. Even when the number of target regions is 7 or more, an individualcan be created similarly as the existing probability of an objectiveprogeny individual in a onetime selected population is 1/64 to 1/16.

When respective target regions are independent respectively withoutlinkage, and are subject to a Mendel's law, how an existing probabilityof an objective progeny individual in an selected population can beappropriately determined in view of the number of progeny individualsobtained by one time mating, and a time until an individual of anobjective cultivar is finally obtained. When the number of progenyindividuals obtained by mating is sufficient, the existing probabilityof an objective progeny individual can be set to be low such as 1/64. Inthis case, a scale of a one time selected population becomes relativelylarge, and a time, the labor and the cost necessary for one timeselection become large, but a cultivar in which the objective number oftarget regions are substituted with a chromosome fragment derived fromthe foreign cultivar can be obtained by relatively small selectiontimes. On the other hand, when the number of progeny individualsobtained by one time mating is small, a scale of the one time selectedpopulation must be reduced. In this case, a time and the cost necessaryfor one time selection can be suppressed, but a selection time isincreased, and a time until an individual of an objective cultivar isfinally obtained is prolonged. For example, as shown in FIG. 11A to FIG.11C, when P4 (ABCD) is created, in the method of FIG. 11A, first, P4(ABCD) is created by three times selection: time of mating P1 (A) and P1(B), and thereafter selecting P2 (AB); time of mating P1 (C) and P1 (D),and thereafter selecting P2 (CD); time of mating P2 (AB) and 22 (CD),and thereafter selecting P4 (ABCD). On the other hand, in the method ofFIG. 11C, P4 (ABCD) is created, for example, by at least five timesselection of: time of mating P1 (A) and P1 (B), and thereafter selectingP2 (AB); time of P1 (C) and P1 (D), and thereafter selecting P2 (CD);time of mating P2 (AB) and P1 (C), and thereafter selecting P3 (ABC);time of mating P1 (B) and 22 (CD), and thereafter selecting P3 (BCD);time of mating P3 (ABC) and P3 (BCD), and thereafter selecting P4(ABCD). In the method of FIG. 11A, a target region substituted with achromosome fragment derived from the foreign cultivar possessed by aparent individual to be mated is different in each parent individual.For this reason, it is necessary to make one time selected populationlarger than the method of FIG. 11C, but P4 (ABCD) can be created by asmaller selection time.

The new cultivar created by the present invention is, as describedabove, a progeny cultivar of a chromosome fragment-substituted line inwhich a part of a chromosome is substituted with a chromosome fragmentderived from the foreign cultivar. In this new cultivar, one or pluraltarget regions are substituted with a chromosome fragment derived fromthe foreign cultivar, and the length of a chromosome fragment iscontrolled by a DNA marker set upstream of the target region, and a DNAmarker set downstream of a target region. In the present invention, byappropriately setting the target region, a useful new cultivar such as anew cultivar described in Examples described later, particularly, ricecultivar, Oryza sativa L. cultivar Koshihikari kazusa 4go can beobtained.

Besides, a new cultivar in which at least one region of plural regionssubstituted with a chromosome fragment derived from foreign cultivarpossessed by a parent individual is substituted with a chromosomefragment derived from the original cultivar can be created. First, anindividual of a cultivar created using the first to fourth methods ofcreating a new cultivar of the present invention, in which two or moretarget regions in a chromosome of the original cultivar are substitutedwith a chromosome fragment derived from the foreign cultivar, or aprogeny individual of this individual, and an individual of the originalcultivar are mated. Then, a progeny individual obtained by this matingis self-mated and, from a progeny individual obtained by thisself-mating, an individual in which at least one region is substitutedwith a chromosome fragment derived from the original cultivar isselected.

For example, the case where an individual P3 (ABC) in which, in achromosome of the original cultivar, all of target regions A, B and Care substituted with a homo-chromosome fragment derived from theoriginal cultivar is created using the first to fourth methods ofcreating a new cultivar of the present invention will be described.First, this P3 (ABC) and an individual of the original cultivar aremated. Then, by self-mating a progeny individual obtained by thismating, an individual P2 (AB) in which only target regions A and B aresubstituted with a homo-chromosome fragment derived from the foreigncultivar and a target region C is substituted with a chromosome fragmentderived from the original cultivar, or an individual P2 (B) in whichonly a target region B is substituted with a homo-chromosome fragmentderived from the foreign cultivar and target regions A and C aresubstituted with a chromosome fragment derived from the originalcultivar is selected. From the foregoing, an individual of a newcultivar in which at least one region of a plurality of regionssubstituted with a chromosome fragment derived from the foreign cultivarpossessed by a parent individual is substituted with a chromosomefragment derived from the original cultivar can be obtained.

EXAMPLES

The following Examples further illustrate the present invention in moredetail, but the present invention is not only limited to the followingExamples.

Example 1

Using the present invention, a new cultivar having improved lodgingresistance of rice cultivar Koshihikari was created.

First, a rice cultivar Habataki which is short, and a rice cultivarKoshihikari were mated, and QTL (Quantitative Trait Locus) analysis wasperformed on a segregated population. As a result, it was seen thatgreat QTL is present in a Sd1 region of the first chromosome. It waspresumed that when the region of Koshihikari is substituted with a generegion derived from Habataki, a height (rod length) of Koshihikaribecomes small, and lodging resistance is strengthened. Then, Habatakiwas backcrossed with Koshihikari to create a chromosomefragment-substituted line in which the Sd1 region of Koshihikari issubstituted with a gene fragment derived from Habataki.

Then, according to the method of plant genome design of the presentinvention, a length of a chromosome fragment region derived fromHabataki of the resulting chromosome-substituted line was regulated todesign a genome. Specifically, a DNA marker SP-4009 in a Sd1 region waslet to be a DNA marker M1 (Sd1), a DNA marker G2003 in a Sdl region waslet to be a DNA marker M2 (Sd1), a DNA marker G2002 in a Sd1 region waslet to be a DNA marker M3 (Sd1), a DNA marker SP-462 in a Sd1 region waslet to be a DNA marker M4 (Sd1), and a DNA marker SP-1259 in a Sd1region was let to be a DNA marker M5 (Sd1). These DNA markers are shownin FIG. 15 and Table 1. A distance d1 between the DNA markers M1 (Sd1)and M2 (Sd1) is about 1.6 kbp, a distance d2 between the DNA markers M2(Sd1) and M4 (Sd1) is about 90 kbp, and a distance d3 between the DNAmakers M4 (Sd1) and M5 (Sd1) is about 750 kbp. Thereby, in a chromosomeof Koshihikari, a length of a Habataki derived chromosome fragment L₁ is90 kbp<L₁<842 kbp.

TABLE 1 Marker Type Position Sequence M1 (Sd1) SP-4009 SNP 38,107,956Upper Sequence: ccgttcatgtgcctgtatggLower Sequence: tgttgcaggaaggtgacaca SP-4009Gc: ttggaaggaacatctagcaggM2 (Sd1) G2003 PCR 38,109,578 TG2003U: cacagcgctcacttctcaTG2003L: tgcaatgtcgtccaccatcg M3 (Sd1) G2002 PCR 38,109,641TG2002U: cacagcgctcacttctca TG2002L: atgatcgtcagcgacagct M4 (Sd1) SP-462SNP 38,199,633 Upper Sequence: aactccagcgtgctaagcLower Sequence: gcattgcatgcaggatcg SP-462Gt: agagcccttcactttcagcM5 (Sd1) SP-1259 SNP 38,949,811 Upper Sequence: aaggctgatgagcactgcLower Sequence: ggcattgtggaagctcttc SP-1259Tc: tctcctttcggagtccc

Then, the resulting chromosome fragment-substituted line and Koshihikariwere mated to harvest 10 progeny individuals (seeds) in which the DNAmarker M3 (Sd1) is a hetero-chromosome region of an allele derived fromKoshihikari and an allele derived from Habataki. All of the resultingseeds were cultivated, and self-fertilized (self-mated) to harvest aseed which is a progeny individual.

This harvested seed was cultivated. After a seedling was grown to suchan extent that the seedling could be transplanted to an agriculturalfield, a DNA was extracted from a leaf of each cultivated individual,and a cultivated individual in which the DNA marker M1 (Sd1) is ahomo-chromosome region of an allele derived from Koshihikari, and DNAmarkers M2 (Sd1) and M3 (Sd1) are a hetero-chromosome region of anallele derived from Koshihikari and an allele derived from Habataki, wasselected.

This selected cultivated individual was self-fertilized (self-mated) toharvest a seed which is a progeny individual. This harvested seed wasfurther cultivated, and a seedling was grown to such an extent that theseedling could be transplanted to an agricultural field, a DNA wasextracted from a leaf of each cultivated individual, and one cultivatedindividual in which DNA markers M1 (Sd1) and M5 (Sd1) are ahomo-chromosome region of an allele derived from Koshihikari, and theDNA markers M2 (Sd1), M3 (Sd1) and M4 (Sd1) are a homo-chromosome regionof an allele derived from Habataki was selected. This selectedcultivated individual is a new cultivar in which a region between theDNA marker M1 (Sd1) and the DNA marker M5 (Sd1) of a Sd1 region ofKoshihikari is substituted with a chromosome fragment derived fromHabataki. The present inventors named this new cultivar as “Koshihikarieichi 4go”.

FIG. 16 is a view schematically showing a genome of Koshihikari eichi4go.

Characters between Koshihikari eichi 4go, Koshihikari and Nihonbare werecompared, and studied (implemented in Aichi Prefecture in 2005 to 2006).Study of characters was performed according to Property Examination forfiling Cultivar Registration based on Plant Cultivar Protection and SeedAct (Year Heisei 10, Law No. 83), Article 5, Section 1. The results areshown in Tables 2 to 4. A rod length of Koshihikari and Nihonbare whichare a control cultivar was 99.0 cm and 86.8 cm, respectively, while arod length of Koshihikari eichi 4go was as short as 83.3 cm. On theother hand, Koshihikari eichi 4go was fundamentally the same asKoshihikari except that a rod length was short, and had a good characterof pre-harvest sprouting difficulty of Koshihikari. Further, as shown inFIG. 17, by a rod length being reduced, lodging resistance was alsoenhanced.

Therefore, from these results, it is clear that, by designing a genomeusing the method of plant genome design of the present invention, andcreating a new cultivar using the method of creating a new cultivar ofthe present invention, a new cultivar having a character of a target canbe created without changing a preferable character possessed by theoriginal cultivar.

TABLE 2 Remark Property value of Value control cultivar measuredKoshihikari Nihonbare in Aichi (Aichi (Aichi Property value of cultivar(comparison with standard cultivar) Prefecture Prefecture, Prefecture,Stage Character 1 2 3 4 5 6 7 8 9 (2005-2006) 2005-2006) 2006) 40 Leaf:Absence Presence 1 1 1 Anthocyanine coloring Leaf: Only tip Only Spot-Whole 1 1 1 Distribution edge like leaf of anthocyanine color Leaf:Absence Presence 1 1 1 Anthocyanine color of auricle 60 Flag leaf: StandHemi- Hori- Recurved 3 3 3 Posture of leaf stand zontal blade (Initialobservation) 90 Flag leaf: Stand Hemi- Hori- Recurved 3 4 4 Posture ofleaf stand zontal blade (Late observation) 55 Heading time ExtremelyEarly Interme- Late 3 3 4 (50% ear early diate August 7 August 7 August17 emergence) 65 Lemma: Absence Pale Interme- Strong Extremely 1 1 1Anthocyanine or diate strong coloring of top extremely part (initialpale observation)

TABLE 3 Property value of cultivar (comparison with standard cultivar)Stage Character 1 2 3 4 5 6 7 8 9 70 Rod: Length Extremely ShortIntermediate Long Extremely (expect for short long ear, expect forfloating rice) Rod: Absence Presence Anthocyanine coloring of knot 72-90Ear: Length Short Intermediate Long of main axis 70 Ear: Ear SmallIntermediate Much number 70-80 Ear: Only tip Only Whole distributionUpper of arista half 60-80 Small ear: Absence or Small Intermediate MuchExtremely Much or less extremely much of trichome small of lemma 80-90Small ear: White Yellow Brown Red Purple Black Color of lemma tip(apiculus color) 90 Ear: Curvature Stand Tilt Hanging Bending extent ofmain axis Ear: Ear type Lanceolate Spindle- Bat- Bloom- Umbellar likelike like Maturing stage Extremely Early Intermediate Late Extremelyearly late Glume color Yellowish Gold Brown Reddish Purple Black whitecolor pale purple Glume color: Absence Golden Brown Purple PurplePattern groove groove spot groove Remark Value Property value of controlcultivar measured Koshihikari Nihonbare in Aichi (Aichi (AichiPrefecture Prefecture, Prefecture, Stage Character (2005-2006)2005-2006) 2006) 70 Rod: Length 4 6 5 (expect for 83.3 cm 99.0 cm 86.8cm ear, expect for floating rice) Rod: 1 1 1 Anthocyanine coloring ofknot 72-90 Ear: Length 4 4 4 of main axis 16.5 cm 16.8 cm 16.4 cm 70Ear: Ear 5 5 5 number  15.3 ears  15.3 ears  12.0 ears 70-80 Ear: 1 1 1distribution of arista 60-80 Small ear: Equivalent Much or less to oftrichome Koshihikari of lemma 80-90 Small ear: 1 1 1 Color of lemma tip(apiculus color) 90 Ear: Curvature 5 5 5 extent of main axis Ear: Eartype 2 2 2 Maturing stage 5 5 6 September 16 September 16 Sep. 28 2007Glume color 1 1 1 Glume color: 1 1 1 Pattern

TABLE 4 Property value of cultivar (comparison with standard cultivar)Stage Character 1 2 3 4 5 6 7 8 9 92 Lemma: Absence or Pale IntermediateDeep Extremely Anthocyanine extremely deep coloring of top pale partLemma: length Short Intermediate Long Lemma: Color Yellowish Gold RedPurple white color Paddy: 1,000 Light Intermediate Heavy particlesweight (mature) Paddy: Phenol Absence Pale Intermediate Deep Presencereaction of palea Brown rice: Length Short Intermediate Long Brown rice:Width Thin Intermediate Thick Brown rice: Circular Half Half Spindle-Long Shape (seen circular spindle- shape spindle- from side) shape shapeBrown rice: Color White Pale Brown Dark Pale red Red Purple Purple Darkbrown spot brown spot purple/ black Brown rice: Absence or Faint StrongFragrances extremely weak Remark Value Property value of controlcultivar measured Koshihikari Nihonbare in Aichi (Aichi (AichiPrefecture Prefecture, Prefecture, Stage Character (2005-2006)2005-2006) 2006) 92 Lemma: 1 1 1 Anthocyanine coloring of top partLemma: length 2 2 2 1.90 mm 1.68 mm 2.01 mm Lemma: Color 1 1 1 Paddy:1,000 5 5 5 particles weight 24.74 g   24.43 g   26.1 g  (mature) Paddy:Phenol 1 1 1 reaction of palea Brown rice: Length 5 5 5 5.03 mm 5.02 mm5.26 mm Brown rice: Width 5 5 5 2.90 mm 2.91 mm 2.88 mm Brown rice: 2 22 Shape (seen 1.85 mm 1.86 mm 2.01 mm from side) Brown rice: Color 2 2 2Brown rice: 1 1 1 Fragrances

Example 2

Using the present invention, a new cultivar in which seeds settingdensity of a rice cultivar Koshihikari was enhanced was created.

First, a rice cultivar Habataki and a rice cultivar Koshihikari weremated, and QTL analysis was performed in a segregated population. As aresult, it became clear that QTL having a higher seeds setting densitythan that of Koshihikari was present in an about 5 Mp region of a firstchromosome. That is, it became clear that a Gn1 gene present in theregion is a causal gene of controlling seeds setting density. Then, itwas predicted that when a Gn1 gene of Koshihikari is substituted with agene region derived from Habataki, seeds setting density of Koshihikariis increased. Then, Habataki was backcrossed with Koshihikari to createa chromosome fragment-substituted line in which a region containing aGn1 gene of Koshihikari is substituted with a gene fragment derived fromHabataki.

Then, according to the method of plant genome design of the presentinvention, a length of a chromosome fragment region derived fromHabataki of the resulting chromosome fragment-substituted line wasregulated to design a genome. Specifically, a DNA marker SP-2032 in aGn1 gene region was let to be a DNA marker M1 (Gn1), a DNA marker SP-170in a Gn1gene region was let to be a DNA marker M2 (Gn1), a DNA markerSP-4028 in a Gn1 gene region was let to be a DNA marker M3 (Gn1), a DNAmarker SP-4038 in a Gn1 gene region was let to be a DNA marker M4 (Gn1),and a DNA marker SP-4030 in a Gn1 gene region was let to be a DNA markerM5 (Gn1). These DNA markers are shown in FIG. 18 and Table 5. A distanced1 between DNA markers M1 (Gn1) and M2 (Gn1) is about 201 kbp, adistance d2 between DNA markers M2 (Gn1) and M4 (Gn1) is about 37 kbp,and a distance d3 between DNA markers M4 (Gn1) and M5 (Gn1) is about 7kbp. Thereby, in a chromosome of Koshihikari, a length of a chromosomefragment L₂ derived from Habataki becomes 37 kbp<L₂<246 kbp.

TABLE 5 Marker Type Position Sequence M1 (Gn1) SP-2032 SNP 5,029,585Upper Sequence: cattgagtccatttcctctgcLower Sequence: gcagctccaagaatgactac SP-2032Tg: attggtgctagagcaactacM2 (Gn1) SP-170 SNP 5,230,897 Upper Sequence: gtgagacatagactatccacLower Sequence: acgcgtacgccacatagac SP-170Ta: agggtgaggaatgtccggtM3 (Gn1) SP 4028 SNP 5,267,633 Upper Sequence: gcagtacctgccttactacgLower Sequence: catttcatgcgagtggtgac SP-4028Ac: tgcacgaatcttggccagagM4 (Gn1) SP-4038 SNP 5,267,932 Upper Sequence: cttaaactcaacttgcacaagtagLower Sequence: actgccgacatgttactgtc SP-4038Gc: gtcccacctgaaacatatccaM5 (Gn1) SP-4030 SNP 5,274,787 Upper Sequence: tctttgattctttggtcgatcgLower Sequence: gcgtacgagagctatagagc SP-4030At: atggatccgtggatcgatcg

Then, the resulting chromosome fragment-substituted line and Koshihikariwas mated, and mating and selection were repeated as in Example 1 toselect an individual of a new cultivar in which a region between a DNAmarker M1 (Gn1) and a DNA marker M5 (Gn1) of a Gn1 gene region ofKoshihikari is substituted with a chromosome fragment derived fromHabataki. The present inventors named this new cultivar as “Koshihikarieichi 2go”. FIG. 19 is a view schematically showing a genome ofKoshihikari eichi 2go.

Characters between Koshihikari eichi 2go, Koshihikari and Nihonbare werecompared and studied as in Example 1 (implemented in Aichi Prefecture in2005 to 2006). The results are shown in Tables 6 to 8. In Tables,“(2005)” indicates a value measured in 2005, and “(2006)” indicates avalue measured in 2006, respectively. Seeds setting density ofKoshihikari which was a control cultivar was 7.01 grains/cm² in 2005,and 8.89 grains/cm² in 2006. Seeds setting density in 2006 of Nihonbarewhich was similarly a control cultivar was 5.99 grains/cm². To thecontrary, Seeds setting density of Koshihikari eichi 2go was 10.7grains/cm² in 2005, and 10.0 grains/cm² in 2006. Like this, Seedssetting density of Koshihikari eichi 2go was very higher and better thanthose of Koshihikari and Nihonbare. On the other hand, no significantdifference was detected between Koshihikari eichi 2go and Koshihikariexcept for high seeds setting density. In addition, also in the casewhere an experiment was implemented in Niigata Prefecture in 2005 usingKoshihikari and Dontokoi as a control cultivar, the approximately sameresults as those of Tables 6 to 8 were obtained.

Therefore, also from these results, it is clear that, by designing agenome using the method of plant genome design of the present invention,and creating a cultivar using the method of creating a new cultivar ofthe present invention, a new cultivar having a target character can becreated without changing a preferable character possessed by theoriginal cultivar.

TABLE 6 Remark Property value of Value control cultivar measuredKoshihikari Nihonbare in Aichi (Aichi (Aichi Property value of cultivar(comparison with standard cultivar) Prefecture Prefecture, Prefecture,Stage Character 1 2 3 4 5 6 7 8 9 (2005-2006) 2005-2006) 2006) 40 Leaf:Absence Pres- 1 1 1 Anthocyanine ence coloring Leaf: Only tip Only Spot-Whole 1 1 1 Distribution of edge like leaf anthocyanine color Leaf:Absence Pres- 1 1 1 Anthocyanine ence color of auricle 60 Flag leaf:Stand Hemi- Hori- Re- 3 3 2 Posture of leaf stand zontal curved blade(Initial observation) 90 Flag leaf: Stand Hemi- Hori- Re- 4 4 4 Postureof leaf stand zontal curved blade (Late observation) 55 Heading timeExtremely Early Interme- Late 3 3 4 (50% ear early diate Aug. 5 2005Aug. 5 2005 August 17 emergence) Aug. 8 2006 Aug. 9 2006 65 Lemma:Absence Pale Interme- Strong Ex- 1 1 1 Anthocyanine or diate tremelycoloring of top extremely strong part (initial pale observation)

TABLE 7 Property value of cultivar (comparison with standard cultivar)Stage Character 1 2 3 4 5 6 7 8 9 70 Rod: Length Extremely ShortIntermediate Long Extremely (expect for short long ear, expect forfloating rice) Rod: Absence Presence Anthocyanine coloring of knot 72-90Ear: Length of Short Intermediate Long main axis 70 Ear: Ear numberSmall Intermediate Much 70-80 Ear: Only tip Only Whole distributionUpper of arista half 60-80 Small ear: Much Absence Small IntermediateMuch Extremely or less of or much trichome of extremely lemma small80-90 Small ear: White Yellow Brown Red Purple Black Color of lemma tip(apiculus color) 90 Ear: Curvature Stand Tilt Hanging Bending extent ofmain axis Ear: Ear type Lanceolate Spindle- Bat- Bloom- Umbellar likelike like Maturing stage Extremely Early Intermediate Late Extremelyearly late Glume color Yellowish Gold Brown Reddish Purple Black whitecolor pale purple Glume color: Absence Golden Brown Purple PurplePattern groove groove spot groove Remark Value Property value or controlcultivar measured Koshihikari Nihonbare in Aichi (Aichi (AichiPrefecture Prefecture, Prefecture, Stage Character (2005-2006)2005-2006) 2006) 70 Rod: Length 6 6 5 (expect for 105.0 cm  108.9 cm 86.8 cm ear, expect for (2005) (2005) floating rice) 95.5 cm 101.0 cm (2006) (2006) Rod: 1 1 1 Anthocyanine coloring of knot 72-90 Ear: Lengthof 4 4 4 main axis 18.0 cm 18.8 cm 16.4 cm (2005) (2005) 15.7 cm 15.1 cm(2006) (2006) 70 Ear: Ear number 5 5 5  17.0 ears  16.8 ears  12.0 ears(2005) (2005)  11.2 ears  13.4 ears (2006) (2006) 70-80 Ear: 1 1 1distribution of arista 60-80 Small ear: Much Equivalent or less of totrichome of Koshihikari lemma 80-90 Small ear: 1 1 1 Color of lemma tip(apiculus color) 90 Ear: Curvature 5 5 5 extent of main axis Ear: Eartype 2 2 2 Maturing stage 5 5 6 Sep. 15 2005 Sep. 18 2005 Sep. 28 2007Sep. 18 2006 Sep. 19 2006 Glume color 1 1 1 Glume color: 1 1 1 Pattern

TABLE 8 Property value of cultivar (comparison with standard cultivar)Stage Character 1 2 3 4 5 6 7 8 9 92 Lemma: Absence or Pale IntermediateDeep Extremely Anthocyanine extremely deep coloring of top pale partLemma: length Short Intermediate Long Lemma: Color Yellowish Gold RedPurple white color Paddy: 1,000 Light Intermediate Heavy particlesweight (mature) Paddy: Phenol Absence Pale Intermediate Deep Presencereaction of palea Brown rice: Length Short Intermediate Long Brown rice:Width Thin Intermediate Thick Brown rice: Shape Circular Half HalfSpindle- Long (seen from side) circular spindle- shape spindle- shapeshape Brown rice: Color White Pale Brown Dark Pale Red Purple PurpleDark brown spot brown red spot purple/ black Brown rice: Absence orFaint Strong Fragrances extremely weak GII Seeds setting Very SparseSlightly Intermediate Slightly Dense Very density sparse sparse densedense Remark Property value of control cultivar Value measuredKoshihikari Nihonbare in Aichi (Aichi (Aichi Prefecture Prefecture,Prefecture, Stage Character (2005-2006) 2005-2006) 2006) 92 Lemma: 1 1 1Anthocyanine coloring of top part Lemma: length 2 2 2 1.83 mm 1.70 mm2.01 mm (2005) (2005) 2.22 mm 1.64 mm (2006) (2006) Lemma: Color 1 1 1Paddy: 1,000 5 5 5 particles weight 24.4 g 24.3 g 26.1 g (mature) (2005)(2005) 25.4 g 24.6 g (2006) (2006) Paddy: Phenol 1 1 1 reaction of paleaBrown rice: Length 5 5 5 4.95 mm 5.03 mm 5.26 mm (2005) (2005) 5.06 mm4.98 mm (2006) (2006) Brown rice: Width 5 5 5 3.01 mm 2.96 mm 2.88 mm(2005) (2005) 2.89 mm 2.86 mm (2006) (2006) Brown rice: Shape 2 2 2(seen from side) 1.98 mm 1.70 mm 2.01 mm (2005) (2005) 2.02 mm 2.03 mm(2006) (2006) Brown rice: Color 2 2 2 Brown rice: 1 1 1 Fragrances GIISeeds setting 7 6 5 density 10.7 grains/cm² 7.01 grains/cm² 5.99grains/cm² (2005) (2005) 10.0 grains/cm² 8.89 grains/cm² (2006) (2006)

Example 3

When Koshihikari is cultivated in Hokkaido, a term from seeding to earemergence is about 144 days and this is too long. That is, when a seedis planted mid-May, ear emergence does not occur until mid-September.However, after mid-September, an air temperature becomes lower inHokkaido, and thereby Koshihikari cannot be normally matured. For thisreason, in order to cultivate Koshihikari in a north district such asHokkaido and the like, conversion into early growth of Koshihikari isnecessary. Then, using the present invention, a new cultivar of a ricecultivar Koshihikari which had been converted into early growth wascreated.

First, a rice cultivar Habataki and a rice cultivar Koshihikari weremated, and QTL analysis was performed in a segregated population. As aresult, QTL which converts Koshihikari into early growth in a tropicalregion was revealed. That is, it was indicated that there was a highpossibility that a Hd1 gene present in the region was a causal gene ofcontrolling conversion into early growth. Then, Habataki was backcrossedwith Koshihikari to create a chromosome fragment-substituted line inwhich a region containing a Hd1 gene of Koshihikari was substituted witha gene fragment derived from Habataki.

Then, according to the method of plant genome design of the presentinvention, a length of a chromosome fragment region derived fromHabataki of the resulting chromosome fragment-substituted line wasregulated to design a genome. Specifically, a DNA marker SP-2513 in theHd1 gene region was let to be a DNA marker M1 (Hd1), a DNA marker SP-586in the Hd1 gene region was let to be a DNA marker M2 (Hd1), a DNA markerSP-2254 in the Hd1 gene region was let to be a DNA marker M3 (Hd1), aDNA marker SP-1603 in the Hd1 gene region was let to be a DNA marker M4(Hd1), and a DNA marker SP-604 in the Hd1 gene region was let to be aDNA marker M5 (Hd1). These DNA markers are shown in FIG. 20 and Table 9.A distance d1 between the DNA markers M1 (Hd1) and M2 (Hd1) is about 344kbp, a distance d2 between the DNA markers M2 (Hd1) and M4 (Hd1) isabout 1,508 kbp, and a distance d3 between the DNA markers M4 (Hd1) andM5 (Hd1) is about 1,279 kbp. Thereby, a length of a chromosome fragmentL₂ derived from Habataki in a chromosome of Koshihikari becomes 1,507kbp<L₂<3,131 kbp.

TABLE 9 Marker Type Position Sequence M1 (Hd1) SP-2513 SNP 8,818,923Upper Sequence: gcgaaaagatgaggatgtacacLower Sequence: ccgtaggcctttgtcaagtg SP-2513Ct: ctttaatggtggcttatgtcM2 (Hd1) SP-586 SNP 9,163,148 Upper Sequence: gctaggacaagcttatttcagcLower Sequence: tcacgccgatcaagaacg SP-586Ca: cataatttatcgccattttcgcatM3 (Hd1) SP-2254 SNP 9,379,247 Upper Sequence: aggcccttgtactggtacLower Sequence: gtacacaatagttggtgcacc SP-2254Cg: catgataaggtactcctggM4 (Hd1) SP-1603 SNP 10,671,067 Upper Sequence: cctagtccctaaagatctcatgLower Sequence: gatagacatgacggagaagtg SP-1603Tc: gggtggtgttatctctagtM5 (Hd1) SP-604 SNP 11,949,717 Upper Sequence: gcgcaaattccttcagtcacLower Sequence: cagtttcaggtggaagacc SP-604Tc: caagtttcttcctctcattttc

Then, the resulting chromosome fragment-substituted line and Koshihikariwere mated to harvest three progeny individuals (seed) in which the DNAmarker M3 is a hetero-chromosome region of an allele derived fromKoshihikari and an allele derived from Habataki. All of the resultingseeds were cultivated, self-fertilized (self-mated), and seeds which area further progeny individual were harvested.

The harvested seeds were further cultivated. After a seedling was grownto such an extent that the seedling could be transplanted to anagricultural field, a DNA was extracted from a leaf of each cultivatedindividual, and a cultivated individual in which the DNA marker M1 (Hd1)is a homo-chromosome region of an allele derived from Koshihikari, andthe DNA markers M2 (Hd1) and M3 (Hd1) are a hetero-chromosome region ofan allele derived from Koshihikari and an allele derived from Habatakiwas selected.

This selected cultivated individual was self-fertilized (self-mated),and seeds which are a further progeny individual were harvested. Theharvested seeds were further cultivated, a seedling was grown to such anextent that it could be transplanted to an agricultural field, a DNA wasextracted from a leaf of each cultivated individual, and one cultivatedindividual in which the DNA markers M1 (Hd1) and M5 (Hd1) are ahomo-chromosome region of an allele derived from Koshihikari, and theDNA markers M2 (Hd1), M3 (Hd1) and M4 (Hd1) are a homo-chromosome regionof an allele derived from Habataki was selected. This selectedcultivated individual is a new cultivar in which a region between theDNA marker M1 (Hd1) and the DNA marker M5 (Hd1) of the Hd1 region ofKoshihikari is substituted with a chromosome fragment derived fromHabataki. The present inventers named this new cultivar as “Koshihikarieichi 3go”.

FIG. 21 is a view schematically showing a genome of Koshihikari eichi3go.

Characters between Koshihikari eichi 3go Koshihikari and Nihonbare werecompared and studied as in Example 1 (implemented in Aichi Prefecture in2005 to 2006). Study results are shown in Tables 10 to 12. In Tables“(2005)” indicates a value measured in 2005, and “(2006)” indicates avalue measured in 2006, respectively. A heading time of Koshihikari andNihonbare which were a control cultivar was August 7 and August 17,respectively, while a heading time of Koshihikari eichi 3go was July 27,being 10 days earlier than those of the control cultivars. In addition,a maturing term of Koshihikari and Nihonbare was September 18 andSeptember 28, respectively, while the maturing term of Koshihikari eichi3go was September 7, and it was seen that as a heading time becomesearlier, a maturing term becomes earlier. In addition, due to an earlierheading time, a rod length of Koshihikari eichi 3go was reduced. On theother hand, other characters of Koshihikari eichi 3go were fundamentallythe same as those of Koshihikari, and Koshihikari eichi 3go also had agood character of pre-harvest sprouting difficulty possessed byKoshihikari.

TABLE 10 Remark Property value of Value control cultivar measuredKoshihikari Nihonbare in Aichi (Aichi (Aichi Property value of cultivar(comparison with standard cultivar) Prefecture Prefecture, Prefecture,Stage Character 1 2 3 4 5 6 7 8 9 (2005-2006) 2005-2006) 2006) 40 Leaf:Absence Pres- 1 1 1 Anthocyanine ence coloring Leaf: Only Only Spot-Whole 1 1 1 Distribution tip edge like leaf of anthocyanine color Leaf:Absence Pres- 1 1 1 Anthocyanine ence color of auricle 60 Flag leaf:Stand Hemi- Hori- Re- 3 3 3 Posture of leaf stand zontal curved blade(Initial observation) 90 Flag leaf: Stand Hemi- Hori- Re- 4 4 4 Postureof leaf stand zontal curved blade (Late observation) 55 Heading time(50% Extremely Early Interme- Late 2 3 4 ear emergence) early diate July27 August 7 August 17 65 Lemma: Absence Pale Interme- Strong Ex- 1 1 1Anthocyanine or diate tremely coloring of top extremely strong part(initial pale observation)

TABLE 11 Property value of cultivar (comparison with standard cultivar)Stage Character 1 2 3 4 5 6 7 8 9 70 Rod: Length Extremely ShortInterme- Long Ex- (expect for short diate tremely ear, expect long forfloating rice) Rod: Absence Pres- Anthocyanine ence coloring of knot72-90 Ear: Length Short Interme- Long of main axis diate 70 Ear: EarSmall Interme- Much number diate 70-80 Ear: Only tip Only Wholedistribution Upper of arista half 60-80 Small ear: Absence or SmallInterme- Much Ex- Much or less extremely diate tremely of trichome smallmuch of lemma 80-90 Small ear: White Yellow Brown Red Purple Black Colorof lemma tip (apiculus color) 90 Ear: Stand Tilt Hanging Bend- Curvatureing extent of main axis Ear: Ear type Lanceolate Spindle- Bat- Bloom-Umbellar like like like Maturing Extremely Early Interme- Late Ex- stageearly diate tremely late Glume color Yellowish Gold Brown Reddish PurpleBlack white color pale purple Glume color: Absence Golden Brown PurplePurple Pattern groove groove spot groove Remark Value Property value ofcontrol cultivar measured Koshihikari Nihonbare in Aichi (Aichi (AichiPrefecture Prefecture, Prefecture, Stage Character (2005-2006)2005-2006) 2006) 70 Rod: Length 4 6 5 (expect for 83.7 cm 105.0 cm  86.8cm ear, expect for floating rice) Rod: 1 1 1 Anthocyanine coloring ofknot 72-90 Ear: Length 4 4 4 of main axis 18.0 cm 17.0 cm 16.4 cm 70Ear: Ear 5 5 5 number  18.0 ears  15.1 ears  12.0 ears 70-80 Ear: 1 1 1distribution of arista 60-80 Small ear: Equivalent Much or less to oftrichome Koshihikari of lemma 80-90 Small ear: 1 1 1 Color of lemma tip(apiculus color) 90 Ear: 5 5 5 Curvature extent of main axis Ear: Eartype 2 2 2 Maturing 4 5 6 stage September 7 September 18 September 28Glume color 1 1 1 Glume color: 1 1 1 Pattern

TABLE 12 Property value of cultivar (comparison with standard cultivar)Stage Character 1 2 3 4 5 6 7 8 9 92 Lemma: Absence or Pale IntermediateDeep Extremely Anthocyanine extremely deep coloring of top pale partLemma: length Short Intermediate Long Lemma: Color Yellowish Gold RedPurple white color Paddy: 1,000 Light Intermediate Heavy particlesweight (mature) Paddy: Phenol Absence Pale Intermediate Deep Presencereaction of palea Brown rice: Length Short Intermediate Long Brown rice:Width Thin Intermediate Thick Brown rice: Shape Circular Half HalfSpindle- Long (seen from side) circular spindle- shape spindle- shapeshape Brown rice: Color White Pale Brown Dark Pale Red Purple PurpleDark brown spot brown red spot purple/ black Brown rice: Absence orFaint Strong Fragrances extremely weak Remark Value Property value ofcontrol cultivar measured Koshihikari Nihonbare in Aichi (Aichi (AichiPrefecture Prefecture, Prefecture, Stage Character (2005-2006)2005-2006) 2006) 92 Lemma: 1 1 1 Anthocyanine coloring of top partLemma: length 2 2 2  1.9 mm 1.67 mm 2.01 mm Lemma: Color 1 1 1 Paddy:1,000 5 5 5 particles weight 26.0 g  24.3 g  26.1 g  (mature) Paddy:Phenol 1 1 1 reaction of palea Brown rice: Length 5 5 5 5.05 mm 5.00 mm5.26 mm Brown rice: Width 5 5 5 2.96 mm 2.91 mm 2.88 mm Brown rice:Shape 2 2 2 (seen from side) 2.05 mm 1.87 mm 2.01 mm Brown rice: Color 22 2 Brown rice: 1 1 1 Fragrances

Actually, Koshihikari eichi 3go was cultivated in Hokkaido and, as aresult, the growth of Koshihikari eichi 3go was earlier than that ofKoshihikari by 24 days. In addition, Koshihikari eichi 3go wasapproximately normally matured unlike Koshihikari. Also from theseresults, it is clear that a new cultivar having a target character canbe created without changing a preferable character possessed by theoriginal cultivar, by designing a genome using the method of plantgenome design of the present invention, and creating a cultivar usingthe method of creating a new cultivar of the present invention.

Thereafter, it was seen that a region containing the Hd1 gene ofHabataki has interesting regulating function on Koshihikari. That is, aregion containing the Hd1 gene of Habataki has the function ofconverting Koshihikari into early growth in a region northerner thanNagoya, but had the function of converting Koshihikari into late growthin a region Southerner than Okinawa. For example, when Koshihikari eichi3go was cultivated in Nagoya, a growth of Koshihikari eichi 3go wasconverted into earlier growth than that of Koshihikari by about 10 days.

On the other hand, Koshihikari eichi 3go was cultivated in South Lawnsenof Ho chi mink in Vietnam having tropical weather and, as a result, thegrowth of Koshihikari eichi 3go was converted into later growth thanthat of Koshihikari by 11 days. That is, it was revealed thatKoshihikari eichi 3go can be cultivated better in both of a north regionand a south region.

Koshihikari is an excellent cultivar good in a taste, but in a northdistrict, a term from seeding to ear emergence is too long, andKoshihikari cannot be safely ear-emerged and matured. Conversely, in asouth district, since Koshihikari has a too short heading time, andcannot take a yield, a cultivating region thereof is limited. Forexample, when Koshihikari is cultivated in a tropical district, earemergence occurs in only about 35 days, and a sufficient yield is notobtained in many cases. To the contrary, Koshihikari eichi 3go createdusing the method of creating a new cultivar of the present invention hasexcellent growth property of a very wide cultivatable region, while agood character of Koshihikari such as a taste and the like is retained.

Example 4

In order to improve yield property and lodging resistance of Koshihikarieichi 3go created in Example 3, a new cultivar Koshihikari kazusa 4gohaving all Habataki-derived chromosome regions possessed by Koshihikarieichi 2go, Koshihikari eichi 3go, and Koshihikari eichi 4go,respectively, was created using the fourth method of the presentinvention.

Specifically, Koshihikari eichi 3go and Koshihikari eichi 2go weremated, two of the resulting progeny individuals (seeds) were cultivated,and these were self-fertilized (self-mated). From progeny individualsobtained by this self-fertilizing, 100 seeds which were a furtherprogeny individual were obtained. All of the 100 seeds were cultivated,DNA markers of respective progeny individuals were investigated, and onecultivated individual in which both of the DNA marker M3 (Hd1) and theDNA marker M3 (Gn1) are a homo-chromosome region of an allele derivedfrom Habataki was selected. This individual was defined as P2 (HG).

According to the DNA markers of respective progeny individuals,respective individuals were grown, a DNA was extracted from a leafsampled from a seedling, and analysis was performed using this DNA.

On the other hand, Koshihikari eichi 4go and Koshihikari eichi 2go weremated, five of the resulting progeny individuals (seeds) werecultivated, and these were self-fertilized (self-mated). From progenyindividuals obtained by this self-fertilizing, 150 seeds which were afurther progeny individual were obtained. All of the 150 seeds werecultivated, and DNA markers of respective progeny individuals wereinvestigated, and one individual in which both of the DNA marker M3(Sd1) and the DNA marker M3 (Gn1) are a homo-chromosome region of anallele derived from Habataki was selected. This individual was definedas P2 (SG).

Then, P2 (HG) and P2 (SG) were mated, and two of the resulting progenyindividuals (seeds) were cultivated, and these were self-fertilized(self-mated). From progeny individuals obtained this self-fertilizing,100 seeds which are a further progeny individual were obtained. All ofthe 100 seeds were cultivated, DNA markers of respective progenyindividuals were investigated, and one individual of a cultivatedindividual in which both of the DNA marker M3 (Hd1) and the DNA markerM3 (Sd1) were a homo-chromosome region of an allele derived fromHabataki was selected. The present inventers named this new cultivar as“Koshihikari kazusa 4go”. FIG. 22 is a view schematically showing agenome of Koshihikari kazusa 4go. In a chromosome of Koshihikari kazusa4go, all of the Hd1 gene region, the Sd1 region and the Gn1 gene regionare substituted with a homo-chromosome fragment derived from Habataki.

Characters between Koshihikari kazusa 4go Koshihikari and Nihonbare werecompared and studied as in Example 1. The results are shown in Tables 13to 16. Koshihikari kazusa 4go had a shorter rod length and higherlodging resistance as compared with Koshihikari and Nihonbare which area control cultivar, like Koshihikari eichi 4go. In addition, likeKoshihikari eichi 3go, a heading time of Koshihikari kazusa 4go wasearlier by 9 days, and a maturing term of Koshihikari kazusa 4go wasearlier than those of Koshihikari and Nihonbare. Further, likeKoshihikari eichi 2go, seeds setting density of Koshihikari kazusa 4gowas higher, and the number of main stem grains of Koshihikari kazusa 4gowas greater than those of Koshihikari and Nihonbare. That is, inKoshihikari kazusa 4go, seeds setting density was higher relative to alength of an ear thereof. In addition, a weight per 1,000 paddies(mature) of Koshihikari kazusa 4go became higher than that ofKoshihikari which was the original cultivar. Particularly, Koshihikarikazusa 4go had a very higher ear harvest coefficient than those ofKoshihikari and Nihonbare, and it was seen that harvesting property ofKoshihikari kazusa 4go was extremely good. On the other hand,Koshihikari kazusa 4go had fundamentally the same other characters asthose of Koshihikari.

TABLE 13 Property value of Property value of cultivar Remark controlcultivar (comparison with standard cultivar) Value Koshi- StageCharacter 1 2 3 4 5 6 7 8 9 measured hikari Nihonbare 40 Leaf: AbsencePres- 1 1 1 Anthocyanine ence coloring Leaf: Only tip Only Spot- Whole 11 1 Distribution edge like leaf of anthocyanine color Leaf: AbsencePres- 1 1 1 Anthocyanine ence color of auricle 60 Flag leaf: Stand Hemi-Hori- Re- 3 3 3 Posture of leaf stand zontal curved blade (Initialobservation) 90 Flag leaf: Stand Hemi- Hori- Re- 4 4 4 Posture of leafstand zontal curved blade (Late observation) 55 Heading time ExtremelyEarly Interme- Late 2 3 4 (50% ear early diate July 30 August 8 August19 emergence) 65 Lemma: Absence Pale Interme- Strong Ex- 1 1 1Anthocyanine or diate tremely coloring of top extremely strong part(initial pale observation)

TABLE 14 Property value of cultivar (comparison with standard cultivar)Stage Character 1 2 3 4 5 6 7 8 9 70 Rod: Length Extremely ShortIntermediate Long Extremely (expect for short long ear, expect forfloating rice) Rod: Absence Presence Anthocyanine coloring of knot 72-90Ear: Length of Short Intermediate Long main axis 70 Ear: Ear numberSmall Intermediate Much 70-80 Ear: Only tip Only Whole distributionUpper of arista half 60-80 Small ear: Absence or Small Intermediate MuchExtremely Much or less extremely much of trichome of small lemma 80-90Small ear: White Yellow Brown Red Purple Black Color of lemma tip(apiculus color) 90 Ear: Curvature Stand Tilt Hanging Bending extent ofmain axis Ear: Ear type Lanceolate Spindle- Bat- Bloom- Umbellar likelike like Maturing stage Extremely Early Intermediate Late Extremelyearly late Glume color Yellowish Gold Brown Reddish Purple Black whitecolor pale purple Glume color: Absence Golden Brown Purple PurplePattern groove groove spot groove Remark Value Property value of controlcultivar Stage Character measured Koshihikari Nihonbare 70 Rod: Length 36 4 (expect for 72.0 cm 105.2 cm  80.2 cm ear, expect for floating rice)Rod: 1 1 1 Anthocyanine coloring of knot 72-90 Ear: Length of 4 4 4 mainaxis 15.1 cm 14.7 cm 14.6 cm 70 Ear: Ear number 5 5 5  13.4 ears  12.4ears   14 ears 70-80 Ear: 1 1 1 distribution of arista 60-80 Small ear:Equivalent Much or less to of trichome of Koshihikari lemma 80-90 Smallear: 1 1 1 Color of lemma tip (apiculus color) 90 Ear: Curvature 5 5 5extent of main axis Ear: Ear type 2 2 2 Maturing stage 4 5 6 September 9September 17 September 29 Glume color 1 1 1 Glume color: 1 1 1 Pattern

TABLE 15 Property value of cultivar (comparison with standard cultivar)Stage Character 1 2 3 4 5 6 7 8 9 92 Lemma: Absence Pale IntermediateDeep Extremely Anthocyanine or deep coloring of top extremely part paleLemma: length Short Intermediate Long Lemma: Color Yellowish Gold RedPurple white color Paddy: 1,000 Light Intermediate Heavy particlesweight (mature) Paddy: Phenol Absence Pale Intermediate Deep Presencereaction of palea Brown rice: Length Short Intermediate Long Brown rice:Width Thin Intermediate Thick Brown rice: Shape Circular Half HalfSpindle- Long (seen from side) circular spindle- shape spindle- shapeshape Brown rice: Color White Pale Brown Dark Pale Red Purple PurpleDark brown spot brown red spot purple/ black Brown rice: Absence FaintStrong Fragrances or extremely weak GII Seeds setting Very SparseSlightly Interme- Slightly Dense Very density sparse sparse diate densedense Remark Value Property value of control cultivar Stage Charactermeasured Koshihikari Nihonbare 92 Lemma: 1 1 1 Anthocyanine coloring oftop part Lemma: length 2 2 2 2.0 mm 1.92 mm 1.90 mm Lemma: Color 1 1 1Paddy: 1,000 6 5 6 particles weight 23.6 g 22.9 g 23.7 g (mature) Paddy:Phenol 1 1 1 reaction of palea Brown rice: Length 5 5 6 5.10 mm 5.09 mm5.26 mm Brown rice: Width 5 5 5 2.85 mm 2.81 mm 2.85 mm Brown rice:Shape 2 2 2 (seen from side) 1.96 mm 1.98 mm 2.03 mm Brown rice: Color 22 2 Brown rice: 1 1 1 Fragrances GII Seeds setting 7 6 5 density 10.5grains/cm² 9.7 grains/cm² 7.8 grains/cm²

TABLE 16 Property value of cultivar Remark Property value of (comparisonwith standard cultivar) Value control cultivar Stage Character 1 2 3 4 56 7 8 9 measured Koshihikari Nihonbare GIII Main stem grain ExtremelySmall Interme- Large Extremely 6 5 4 number small diate large 157 grains143 grains 113 grains Main stem first Extremely Short Interme- LongExtremely 8 8 8 internode length short diate long 33.3 cm 36.54 cm 34.7cm Main stem second Extremely Short Interme- Long Extremely 5 7 5internode length short diate long 18.8 cm 21.42 cm 17.35 cm Main stemthird Extremely Short Interme- Long Extremely 4 7 5 internode lengthshort diate long 12.22 cm 18.74 cm 14.35 cm Main stem forth ExtremelyShort Interme- Long Extremely 3 7 5 internode length short diate long7.26 cm 14.9 cm 8.375 cm Main stem fifth Extremely Short Interme- LongExtremely 3 7 5 internode length short diate long 1.72 cm 8.24 cm 3.85cm Main stem sixth Extremely Short Interme- Long Extremely 1 5 2internode length short diate long 0 cm 2.9 cm 1 cm Thick of mainExtremely Thin Interme- Thick Extremely 5 5 5 stem paddy thin diatethick 2.17 mm 2.17 mm 2.21 mm Length of main Extremely Short Interme-Long Extremely 3 3 3 stem paddy short diate long 7.4 mm 7.13 mm 7.53 mmWidth of main Extremely Narrow Interme- Wide Extremely 5 5 5 stem paddynarrow diate wide 3.36 mm 3.26 mm 3.27 mm Ear harvest Extremely LowInterme- High Extremely 8 5 2 coefficient low diate high 56.8 44.6 36.9

That is, by comparison of characters between Koshihikari kazusa 4go,Koshihikari and Nihonbare, it was confirmed that Koshihikari kazusa 4gohas a character which was expected at genome design that the Sd1 gene,the Hd1 gene and the Gn1 gene are substituted with Habataki-derivedgenes without influencing on other characters of Koshihikari as theoriginal cultivar.

Therefore, from these results, it is clear that, by mating new cultivarscreated by using the method of creating a new cultivar of the presentinvention, a progeny individual in which all of homo-chromosomefragments derived from the foreign cultivar possessed by the seed parentand the pollen parent, respectively, are accumulated can be obtained,and is also clear that a new cultivar having plural kinds of targetcharacters can be created without changing a preferable characterpossessed by the original cultivar.

Then, a Koshihikari genome substitution rate (ratio of Koshihikarigenome as original cultivar occupied in entire genome) regarding eachcultivar of Koshihikari kazusa 4go and Koshihikari eiichi 2 to 4gocreated in Examples is shown.

Lengths of chromosome fragments derived from Habataki in Koshihikarieiichi 2 to 4go created in Examples 1 to 3 are shown in Table 17. Apossible minimum length of a Habataki-derived chromosome fragment is d2and a maximum length thereof is d1+d2+d3.

TABLE 17 Distance between markers Minimum Maximum (kbp) (kbp) (kbp) d1d2 d3 d2 d1 + d2 + d3 Koshihikari 201.3 37.0 6.9 37.0 245.2 eiichi 2goKoshihikari 344.2 1507.9 1278.7 1507.9 3130.8 eiichi 3go Koshihikari 1.690.4 750.2 90.4 841.9 eiichi 4go

From a length of Habataki-derived chromosome fragments in Table 17, aforeign genome substitution rate [Habataki-derived chromosome fragmentlength/entire genome length×100(%)] and a Koshihikari genomesubstitution rate [100%−foreign genome substitution rate] werecalculated. Results are shown in Table 18. A full genome length was letto be 430 Mbp.

TABLE 18 Foreign genome Koshihikari genome substitution rate (%)substitution rate (%) Maximum Minimum Maximum Minimum Koshihikari 0.05700.0086 99.9914 99.9430 eiichi 2go Koshihikari 0.7281 0.3507 99.649399.2719 eiichi 3go Koshihikari 0.1958 0.0210 99.9790 99.8042 eiichi 4goKoshihikari 0.9809 0.3803 99.6197 99.0191 kazusa 4go

As shown in Table 18, since all of new cultivars created by the methodof the present invention have a sufficiently high Koshihikari genomesubstitution rate (due to sufficiently small rate of substitution withforeign gene), characters other than a character due to a recombinedobjective gene are equivalent to those of Koshihikari (originalcultivar), and it can be said that the new cultivars are an isogenicline of Koshihikari.

Koshihikari kazusa 4go is a new cultivar created by using the method ofcreating a new cultivar of the present invention, and is a veryexcellent new cultivar which is excellent in lodging resistance, and hasa great yield and a wide cultivation region while a good character suchas a taste possessed by Koshihikari is maintained. Then, the applicantdeposited (accepted date: Jul. 1, 2008) Koshihikari kazusa 4go as anovel plant in International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (Tsukuba CenterChuou 6^(th), Higashi 1-1-1, Tsukuba-shi, Ibaragi-ken, Japan (Post code305-8566)), and this was transferred to International Deposition in thesame Institute (acceptance date: Jun. 30, 2009). An accession number ofInternational Deposition is FERM AMP-11140.

INDUSTRIAL APPLICABILITY

Since by using the method of creating a new cultivar of the presentinvention, a region of a chromosome fragment derived from the foreigncultivar to be introduced can be controlled, and a new cultivar havingone or plural kinds of target characters can be made without changing apreferable character possessed by the original cultivar, the method canbe utilized, particularly, in the field of plant breeding.

1-23. (canceled)
 24. A method of discriminating a plant cultivar whetheror not a plant individual is a particular cultivar, the methodcomprising: letting SP-4009 to be a DNA marker M1, letting G2003 to be aDNA marker M2, letting G2002 to be a DNA marker M3, letting SP-462 to bea DNA marker M4, and letting SP-1259 to be a DNA marker M5; typing oneor more of the DNA markers selected from the group consisting of the DNAmarkers M1 to M5 by genome analysis of the plant individual; anddiscriminating that the plant individual is Oryza sativa L. cultivarKoshihikari eichi 4go or Oryza sativa L. cultivar Koshihikari kazusa 4gowhen the resulting typing result is consistent with the result of Oryzasativa L. cultivar Koshihikari eichi 4go or Oryza sativa L. cultivarKoshihikari kazusa 4go.
 25. A method of discriminating whether or not aplant individual is a particular cultivar, the method comprising:letting SP-2032 to be a DNA marker M1, letting SP-170 to be a DNA markerM2, letting SP-4028 to be a DNA marker M3, letting SP4038 to be a DNAmarker M4, and letting SP-4030 to be a DNA marker M5; typing one or moreof the DNA markers selected from the group consisting of the DNA markersM1 to M5 by genome analysis of the plant individual; and discriminatingthat the plant individual is Oryza sativa L. cultivar Koshihikari eichi2go or Oryza sativa L. cultivar Koshihikari kazusa 4go when theresulting typing result is consistent with the result of Oryza sativa L.cultivar Koshihikari eichi 2go or Oryza sativa L. cultivar Koshihikarikazusa 4go.
 26. A method of discriminating a plant cultivar of thepresent invention is the method for discriminating whether or not aplant individual is a particular cultivar, the method comprising:letting P-2513 to be a DNA marker M1, letting SP-586 to be a DNA markerM2, letting SP-2254 to be a DNA marker M3, letting SP-1603 to be a DNAmarker M4, and letting SP-604 to be a DNA marker M5: typing one or moreof the DNA markers selected from the group consisting of the DNA markersM1 to M5 by genome analysis of the plant individual; and discriminatingthat the plant individual is Oryza sativa L. cultivar Koshihikari eichi3go or Oryza sativa L. cultivar Koshihikari kazusa 4go when theresulting typing result is consistent with the result of Oryza sativa L.cultivar Koshihikari eichi 3go or Oryza sativa L. cultivar Koshihikarikazusa 4go.